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Modern Experimental Biochemistry [3 ed.]
 0805331115, 9780805331110

Table of contents :
Front Cover
Of Related Interest
Title Page
Copyright Page
Preface
Acknowledgments
Table of Contents
Part One. Theory and Experimental Techniques
CHAPTER 1. Introduction to the Biochemistry Laboratory
A. Safety in the Laboratory
B. The Laboratory Notebook and Experiment Reports
Details of Experimental Write-up
C. Cleaning Laboratory Glassware
Glassware
Quartz and Glass Cuvettes
D. Preparation and Storage of Solutions
Water Quality
Solution Preparation
E. Quantitative Transfer of Liquids
Filling a Pipet
Disposable Pasteur Pipets
Calibrated Pipets
Automatic Pipetting Systems
Cleaning and Drying Pipets
F. Statistical Analysis of Experimental Data
Analysis of Experimental Data
Determination of the Mean, Sample Deviation, and Standard Deviation
Statistical Analysis in Practice
Study Problems
Further Reading
On the Web
CHAPTER 2. General Laboratory Procedures
A. pH, Buffers, Electrodes, and Biosensors
Measurement of pH 3
Biochemical Buffers
Selection of a Biochemical Buffer
The Oxygen Electrode
Ion-Selective Electrodes and Biosensors
B. Measurement of Protein Solutions
The Biuret and Lowry Assays
The Bradford Assay
The BCA Assay
The Spectrophotometric Assay
C. Measurement of Nucleic Acid Solutions
The Spectrophotometric Assay
Other Assays for Nucleic Acids
D. Techniques for Sample Preparation
Dialysis
Ultrafiltration
Lyophilization and Centrifugal Vacuum Concentration 5
Study Problems
Further Reading
On the Web
CHAPTER 3. Purification and Identification of Biomolecules by Chromatography
A. Introduction to Chromatography
Partition versus Adsorption Chromatography
B. Planar Chromatography (Paper and Thin-Layer)
Preparation of the Stationary Support 6
Solvent Development of the Support
Detection and Measurement of Components
Applications of Planar Chromatography
C. Gas Chromatography (GC)
Instrumentation
Selection of Operating Conditions
Analysis of GC Data
Advantages and Limitations of GC
D. Column Chromatography
Operation of a Chromatographic Column
Packing the Column
Loading the Column
Eluting the Column
Collecting the Eluent
Detection of Eluting Components
E. Ion-Exchange Chromatography
Ion-Exchange Resins
Selection of the Ion Exchanger
Choice of Buffer
Preparation of the Ion Exchanger
Using the Ion-Exchange Resin
Storage of Resins
F. Gel Exclusion Chromatography
Theory of Gel Filtration
Physical Characterization of Gel Chromatography
Chemical Properties of Gels
Selecting a Gel
Gel Preparation and Storage
Operation of a Gel Column
Applications of Gel Exclusion Chromatography
G. High-Performance Liquid Chromatography (HPLC)
Instrumentation
Stationary Phases in HPLC
The Mobile Phase
Solvents for HPLC Operation
Gradient Elution in HPLC
Sample Preparation and Selection of HPLC Operating Conditions
FPLC—A Modification of HPLC 9
H. Affinity Chromatography and Immunoadsorption
Chromatographic Media
The Immobilized Ligand
Attachment of Ligand to Matrix
Experimental Procedure for Affinity Chromatography
I. Perfusion Chromatography
Study Problems
Further Reading
Chromatography on the Web
CHAPTER 4. Characterization of Proteins and Nucleic Acids by Electrophoresis
A. Theory of Electrophoresis
B. Methods of Electrophoresis
Polyacrylamide Gel Electrophoresis (PAGE)
Nucleic Acid Sequencing Gels
Agarose Gel Electrophoresis
Pulsed Field Gel Electrophoresis (PFGE)
Isoelectric Focusing of Proteins
Two-Dimensional Electrophoresis of Proteins
Capillary Electrophoresis (CE)
Immunoelectrophoresis (IE)
C. Practical Aspects of Electrophoresis
Instrumentation
Reagents
Staining and Detecting Electrophoresis Bands
Protein and Nucleic Acid Blotting
Analysis of Electrophoresis Results 1
Study Problems
Further Reading
Electrophoresis on the Web
CHAPTER 5. Spectroscopic Analysis of Biomolecules
A. Ultraviolet-Visible Absorption Spectrophotometry Principles
Instrumentation
Applications
B. Fluorescence Spectrophotometry
Principles
Instrumentation
Applications
Difficulties in Fluorescence Measurements
C. Other Spectroscopic Methods
Nuclear Magnetic Resonance Spectroscopy
Mass Spectrometry
Study Problems
Further Reading
Spectroscopy on the Web
CHAPTER 6. Radioisotopes in Biochemical Research
A. Origin and Properties of Radioactivity
Introduction
Isotopes in Biochemistry
Units of Radioactivity
B. Detection and Measurement of Radioactivity
Liquid Scintillation Counting
Geiger-Muller Counting of Radioactivity
Scintillation Counting of y Rays
Background Radiation
Applications of Radioisotopes
Statistical Analysis of Radioactivity Measurements
C. Radioisotopes and Safety
Preparation for the Experiment
Performing the Experiment
Study Problems
Further Reading
Radioisotopes on the Web
CHAPTER 7. Centrifugation in Biochemical Research
A. Basic Principles of Centrifugation
B. Instrumentation for Centrifugation
Low-Speed Centrifuges
High-Speed Centrifuges
Ultracentrifuges
C. Applications of Centrifugation
Preparative Techniques
Analytical Measurements
Care of Centrifuges and Rotors
Study Problems
Further Reading
Centrifugation on the Web
Part Two. Experiments
EXPERIMENT 1. Using the Computer in Biochemical Research
EXPERIMENT 2. Structural Analysis of a Dipeptide
EXPERIMENT 3. Using Gel Filtration to Study Ligand-Protein Interactions
EXPERIMENT 4. Isolation and Characterization of Bovine Milk a-Lactalbumin
EXPERIMENT 5. Kinetic Analysis of Tyrosinase
EXPERIMENT 6. Purification and Characterization of Triacylglycerols in Natural Oils
EXPERIMENT 7. Identification of Serum Glycoproteins by SDS-PAGE and Western Blotting
EXPERIMENT 8. Isolation and Characterization of Plant Pigments
EXPERIMENT 9. Photoinduced Proton Transport through Chloroplast Membranes
EXPERIMENT 10. Isolation, Subfractionation, and Enzymatic Analysis of Beef Heart Mitochondria
EXPERIMENT 11. Measurement of Cholesterol and Vitamin C in Biological Samples
EXPERIMENT 12. Activity and Thermal Stability of Gel-immobilized Peroxidase
EXPERIMENT 13. Extraction and Characterization of Bacterial DNA
EXPERIMENT 14. Plasmid DNA Isolation and Characterization by Electrophoresis
EXPERIMENT 15. The Action of Restriction Endonucleases on Plasmid or Viral DNA
Appendices
APPENDIX I. Properties of Common Acids and Bases
APPENDIX II. Properties of Common Buffer Compounds
APPENDIX III. pKa Values and pH, Values of Amino Acids
APPENDIX IV. Molecular Weights of Some Common Proteins
APPENDIX V. Common Abbreviations Used in This Text
APPENDIX VI. Units of Measurement
APPENDIX VII. Table of the Elements
APPENDIX VIII. Values of t for Analysis of Statistical Confidence Limits
APPENDIX IX. Answers to Selected Study Problems
Index

Citation preview

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Modern

Experimental

Biochemistry

Third

Edition

Rodney Boyer Hope College

An imprint of Addison Wesley Longman San Francisco • Boston • New York

Capetown • Hong Kong • London • Madrid • Mexico City Montreal • Munich • Paris • Singapore • Sydney • Tokyo • Toronto

Disclaimer

The experiments in this book have been exhaustively tested for safety and all attempts have been made to use the least hazardous chemicals and procedures possible. However, the author and publisher cannot be held liable for any injury or damage which may occur during the performance of the experiments. It is assumed that before an experiment is initiated, a Material Safety Data Sheet (MSDS) for each chemical used will have been studied by the instructor and students to ensure its safe handling and disposal. Sponsoring Editor

Ben Roberts

Publishing Associate Production Editor

Jean Lake

Cover Design

Emi Koike

Claudia Herman

Cover Illustration

Quade Paul, FiVth.com

Text Design

Robert Gray/Synapse Creative

Copy Editor Composition and Illustrations Printing and Binding Cover Printer

Mary Prescott Pre-Press Company, Inc. Maple-Vail Press Lehigh Press

Copyright © 2000 by Addison Wesley Longman, Inc. All rights reserved. No part of this publication may be reproduced, stored in a database or retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the publisher. Printed in the United States of America. Published simultaneously in Canada. Library of Congress Cataloging-in-Publication Data Boyer, Rodney Modern experimental biochemistry / Rodney Boyer. - 3rd ed. p. cm.

Includes bibliographical references and index. ISBN 0-8053-3111-5

1. Biochemistry-Methodology. 2. Biochemistry-Laboratory manuals. 3. Molecular biology-Methodology. 4. Molecular biology-Laboratory manuals. I. Title. II. Series. QP519.7.B68 2000 572'.028-dc21 00-044528

23456789 10-MV-04 03 02 01 00

Benjamin/Cummings, an imprint of Addison Wesley Longman 1301 Sansome Street, San Francisco, CA 94111

The undergraduate biochemistry teaching laboratory has become an essential feature in the training of students majoring in biochemistry, chemistry, molecular biology, and related biological sciences. Students training for careers in chemistry and the molecular life sciences must acquire extensive experience working with biomolecules in the laboratory, and a formal laboratory course is usually the first step to gain that experience. This step prepares students for participation in future research and development projects. The purpose of the third edition of this book is to provide junior-senior science students with a modern, balanced, and thorough practice in experimental biochemistry and molecular biology. With such a broad array of potential topics and techniques available, it is difficult to select those that students should experience and master. It is a certainty that one instructor's list of essential topics and techniques to include in a biochemistry teaching lab would vary from another instructor's list. However, there are techniques and concepts that most of us would agree form a "core" in biochemistry laboratory. In selecting experiments for this book I have used my judgment developed from over 25 years of teaching and research and also advice from the Committee on Professional Training (CPT) of the American Chemical Society (ACS). In the Fall 1998 issue of the CPT Newsletter, the core topics of biochemistry suggested for classroom and lab were: Biological Structures and Interactions that Stabilize Biological Molecules. Fundamental building blocks (amino acids, carbohydrates, lipids, nucleotides), organic and inorganic prosthetic groups, biopolymers (nucleic acids, peptides/ proteins, polysaccharides), membranes.

Biological Reactions. Biosynthesis and catabolism of biological molecules (amino acids, carbohydrates, lipids, nucleic acids, peptides/proteins), metabolic cycles, biological catalysis and kinetics, mechanisms, organic and inorganic cofactors.

Biological Equilibria and Energetics. pH/buffers, binding/recognition, proton and electron transport, oxidation/reduction, macromolecular conformations. Some ofthese topics may be covered in laboratory courses. The experiments that are usedfor this purpose should emphasize techniques including error

and statistical analysis of experimental data, spectroscopic methods, electrophoretic techniques, chromatographic separations, isolation and identification ofmacro-molecules.

All of the topics and techniques are included in this edition of the text. As with the first two editions, the book is organized into two parts: I. Theory and Experimental Techniques and II. Experiments. Part I introduces students to theoretical and background material for the experiments. This part may also serve as a supplement for instructors who use their own experiments. In Part II there are 15 experiments that represent all areas of biochemistry, including working with proteins and nucleic acid isolation and characterization. The number of experiments has been reduced from earlier editions at the request of instructors and students who believed the book had more experiments than needed for a typical one-semester course. There are, however, still sufficient experiments for a two-semester course sequence. The reduction in the number of experiments has also been achieved by combining some experiments. Many other changes have been made in the new edition. Approximately one-third of the experiments are entirely new, covering topics such as the use of the Internet in literature/structure searching, Western blotting, ligand-protein interactions, and analysis of amino acids by HPLC or CE. The remaining experiments have been thoroughly revised and updated in written directions and experimental methods. Study problems for student practice are now included at the end of each chapter in Part I as well as in each experiment. Each of the 7 chapters and 15 experiments has 10 study problems, many of which have answers in the Appendix. In addition, the list of literature references at the end of chapters and experiments contains World Wide Web sites for student and instructor use.

Both Parts I and II have been completely rewritten and reflect the many advances in biochemistry-molecular biology theory and techniques. Especially noteworthy have been the technical advances in chromatography (perfusion, FPLC, bioaffinity), electrophoresis (pulsed gel, capillary, nucleic acid sequencing), spectrophotometry (nmr, ms, and diode array detectors), and molecular biology (microsequencing of proteins and nucleic acids, blotting, restriction enzymes). In the development of an experiment, primary consideration was given to the use of modern procedures and techniques. Therefore, students will learn procedures that are now used in actual laboratory settings, whether academic or industrial. Each experiment presents a challenging laboratory situation or problem to be solved by the student. In each experiment, a technique is introduced that allows students to obtain and evaluate properties of a biomolecule, a biochemical reaction, or a biological process. The outline for each experiment is as follows. Introduction and Theory. In this section it is assumed that a student has studied the general subject in the classroom. Only a summary or review of

Preface

ix

significant aspects of background is included. The general discussion includes theoretical and practical information that is generally not available in biochemistry textbooks. The general thrust of the experiment is explained, and a flowchart of the experiment is often presented if appropriate. This outline of the experiment allows the student to recognize the importance of each part of the experiment to the achievement of the overall objective.

Materials and Supplies. A complete list of all materials, supplies, and equipment required for the experiment is provided. An Instructor's Manual describing the preparation of all reagents and solutions and advice on how to set up a biochemistry laboratory is available from the publisher. Experimental Procedure. This section contains a step-by-step detailed procedure for the experiment. It is divided into logical parts for ease of completion and to facilitate the interruption of an experiment, if necessary. To enhance student awareness, an icon (£J) is used to alert students and instructors to possible safety concerns. Analysis of Results. In this section the student is instructed in the proper collection and handling of data from the experiment. Each table or graph that is to be constructed is explained and sample calculations are outlined. Typical data for the experiment may be disclosed but only to aid the student in interpretation of results.

Study Problems. Ten study problems are provided for student practice at the end of each experiment. Some questions will deal with various details of the experiment and numerical problems are emphasized. This symbol (Q) indicates questions that are answered in Appendix IX.

Further Reading. Each experiment ends with a complete list of references that provide either a more detailed theoretical background or an expanded explanation of procedures and techniques. In addition, each chapter and experiment now has Web sites related to topics and techniques.

Acknowledgments

Writing and publishing this textbook required the assistance of many creative, talented, and dedicated individuals. Ben Roberts, Senior Chemistry Editor at Benjamin/Cummings, guided the project through all the necessary steps of writing, reviewing, and publishing. Claudia Herman, Publishing Associate, skillfully assisted in these efforts. I am especially indebted to my Production Editor, Jean Lake, for her constant diligence, patience, and encouragement during the publication stage. I also thank Mrs. Norma Plasman (Hope College), who with her typical efficiency and skill, typed many drafts of the manuscript. Developing a textbook in a rapidly evolving discipline such as biochemistry requires the input of knowledgeable scientists and dedicated teachers. These qualities were present in reviewers of the manuscript including: First Edition

Hugh Akers, Lamar University

John Cronin, Arizona State University

Patricia Dwyer-Hallquist, Appleton Paper Inc. Robert Lindquist, San Francisco State University Kenneth Marx, Dartmouth College Richard Paselk, Humboldt State University William Scovell, Bowling Green State University Ev Trip, University of British Columbia Dennis Vance, University of British Columbia Ronald Watanabe, San Jose State University Second Edition

Larry Byers, Tulane University Stephen Dahms, San Diego State University Anthony Gawienowski, University of Massachusetts, Amherst

Rebecca Jurtshuk, University of Houston Robert Lindquist, San Francisco State University

Denise Magnuson, Texas A&M University Henry Mariani, University of Massachusetts, Boston William Meyer, University of Vermont Ilka Nemere, University of California, Riverside Robert Sanders, University of Kansas

Anthony Toste, Southwest Missouri State University

Steven Wietstock, Alma College Third Edition

Linda Brunauer, Santa Clara University

Jeffrey A. Cohlberg, California State University, Long Beach Diane Dottavio, University of Houston Donald Fox, University of Houston Colleen Johnson, New Mexico State University Robert D. Kuchta, University of Colorado at Boulder Robert Lindquist, San Francisco State University Ray Lutgring, University of Evansville Chris Makaroff, Miami University Susan Martinis, University of Houston Douglas D. McAbee, California State University, Long Beach Linda Roberts, California State University, Sacramento Tim Sherwood, Arkansas Tech University Alexander Volkov, Oakwood College Kimberly Waldron, Regis University William R. Widger, University of Houston

I also thank my wife, Christel, who patiently tolerated the lifestyle changes associated with writing a book. In addition, she designed experimental procedures and searched for ideal laboratory conditions for several experiments. I am happy to report that Mausi, our blue-point Himalayan, is still napping under the desk lamp. I encourage all users of this book to send comments that will assist in the preparation of future editions. Rodney Boyer [email protected]

Table

of

Contents

Part

CHAPTER 1

One

Theory

and

Experimental

Introduction to the Biochemistry Laboratory

3

A.

Safety in the Laboratory

B.

The Laboratory Notebook and Experiment Reports

4

Details of Experimental Write-up

C.

11

Preparation and Storage of Solutions Water Quality

11

Quantitative Transfer of Liquids Filling a Pipet

13

13

Disposable Pasteur Pipets Calibrated Pipets

14

14

Automatic Pipetting Systems

Cleaning and Drying Pipets

F.

11

11

Solution Preparation

E.

10

10

Quartz and Glass Cuvettes

D.

8

9

Cleaning Laboratory Glassware Glassware

Techniques

16

18

Statistical Analysis of Experimental Data Analysis of Experimental Data

18

19

Determination of the Mean, Sample Deviation, and Standard Deviation Statistical Analysis in Practice Study Problems

25

Further Reading On the Web

CHAPTER 2

27 28

General Laboratory Procedures

A.

20

23

29

pH, Buffers, Electrodes, and Biosensors Measurement of pH

30

Biochemical Buffers

32

29

xiii

Selection of a Biochemical Buffer

The Oxygen Electrode

34

37

Ion-Selective Electrodes and Biosensors

B.

Measurement of Protein Solutions

The Biuret and Lowry Assays The Bradford Assay 44

The Spectrophotometric Assay

D.

41

41

43

The BCA Assay

C.

39

44

Measurement of Nucleic Acid Solutions

The Spectrophotometric Assay

46

Other Assays for Nucleic Acids

46

46

Techniques for Sample Preparation Dialysis

48

48

Ultrafiltration

49

Lyophilization and Centrifugal Vacuum Concentration Study Problems

55

Further Reading On the Web

CHAPTER 3

52

56 57

Purification and Identification of Biomolecules by Chromatography

A.

Introduction to Chromatography

59

Partition versus Adsorption Chromatography

B.

60

Planar Chromatography (Paper and Thin-Layer) Preparation of the Stationary Support

62

Solvent Development of the Support

63

Detection and Measurement of Components

Applications of Planar Chromatography

C.

Gas Chromatography (GC) Instrumentation

65

65

Selection of Operating Conditions

Analysis of GC Data

68

68

Advantages and Limitations of GC

D.

Column Chromatography

69

70

Operation of a Chromatographic Column Packing the Column Loading the Column Eluting the Column Collecting the Eluent

72 72 73 73

Ion-Exchange Chromatography Ion-Exchange Resins

71

71

Detection of Eluting Components

E.

64

64

74

75

Selection of the Ion Exchanger

76

61

59

Contents

XV

Choice of Buffer

78

Preparation of the Ion Exchanger

78

Using the Ion-Exchange Resin Storage of Resins

F.

78

79

Gel Exclusion Chromatography Theory of Gel Filtration

79

79

Physical Characterization of Gel Chromatography Chemical Properties of Gels

Selecting a Gel

80

81

83

Gel Preparation and Storage Operation of a Gel Column

83 84

Applications of Gel Exclusion Chromatography

G.

85

High-Performance Liquid Chromatography (HPLC) Instrumentation

87

89

Stationary Phases in HPLC The Mobile Phase

92

95

Solvents for HPLC Operation Gradient Elution in HPLC

96 97

Sample Preparation and Selection of HPLC Operating Conditions FPLC—A Modification of HPLC

H.

98

Affinity Chromatography and Immunoadsorption Chromatographic Media The Immobilized Ligand

101

Attachment of Ligand to Matrix

101

Experimental Procedure for Affinity Chromatography

I.

Perfusion Chromatography

Study Problems

107

Further Reading

108

Chromatography on the Web

CHAPTER 4

104

106

109

Characterization of Proteins and Nucleic Acids by Electrophoresis

111

A.

Theory of Electrophoresis

B.

Methods of Electrophoresis

111 113

Polyacrylamide Gel Electrophoresis (PAGE) Nucleic Acid Sequencing Gels Agarose Gel Electrophoresis

122

Isoelectric Focusing of Proteins

126

127

Two-Dimensional Electrophoresis of Proteins Immunoelectrophoresis (IE)

113

121

Pulsed Field Gel Electrophoresis (PFGE)

Capillary Electrophoresis (CE)

99

100

130 132

130

98

C.

Practical Aspects of Electrophoresis Instrumentation

Reagents

133

133

133

Staining and Detecting Electrophoresis Bands

Protein and Nucleic Acid Blotting Analysis of Electrophoresis Results Study Problems

138

Further Reading

139

Electrophoresis on the Web

CHAPTER 5

1 37

1 40 141

Spectroscopic Analysis of Biomolecules

A.

Ultraviolet-Visible Absorption Spectrophotometry Principles Instrumentation

146

150

Fluorescence Spectrophotometry Principles

157

157

Instrumentation

160

Applications

161

Difficulties in Fluorescence Measurements

C.

Other Spectroscopic Methods

162

163

Nuclear Magnetic Resonance Spectroscopy Mass Spectrometry

Study Problems

168

Further Reading

169 170 171

Radioisotopes in Biochemical Research

A.

Origin and Properties of Radioactivity Introduction

171

171

Isotopes in Biochemistry Units of Radioactivity

B.

163

167

Spectroscopy on the Web

CHAPTER 6

174 175

Detection and Measurement of Radioactivity Liquid Scintillation Counting

Geiger-Muller Counting of Radioactivity Background Radiation

181

182

183

Applications of Radioisotopes

183

Statistical Analysis of Radioactivity Measurements

Radioisotopes and Safety

184

Preparation for the Experiment Performing the Experiment

176

176

Scintillation Counting of y Rays

C.

142

142

Applications

B.

134

136

185 186

184

xvii

Contents

Study Problems

187

Further Reading

187

Radioisotopes on the Web

CHAPTER 7

188

Centrifugation in Biochemical Research

189

A.

Basic Principles of Centrifugation

189

B.

Instrumentation for Centrifugation Low-Speed Centrifuges High-Speed Centrifuges Ultracentrifuges

C.

193

193 195

198

Applications of Centrifugation Preparative Techniques

Analytical Measurements

202

Care of Centrifuges and Rotors Study Problems

207

Further Reading

208

Centrifugation on the Web

Part

Two

201

201

206

208

Experiments

EXPERIMENT 1

Using the Computer in Biochemical Research

EXPERIMENT 2

Structural Analysis of a Dipeptide

EXPERIMENT 3

Using Gel Filtration to Study Ligand-Protein Interactions

EXPERIMENT 4

Isolation and Characterization of Bovine Milk a-Lactalbumin

EXPERIMENT 5

Kinetic Analysis of Tyrosinase

EXPERIMENT 6

Purification and Characterization of Triacylglycerols in Natural Oils

EXPERIMENT 7

EXPERIMENT 9

227

279

303

Identification of Serum Glycoproteins by SDS-PAGE and Western Blotting

EXPERIMENT 8

211

321

Isolation and Characterization of Plant Pigments Photoinduced Proton Transport through Chloroplast Membranes

345

333

243 257

EXPERIMENT 10

Isolation, Subfractionation, and Enzymatic Analysis of Beef Heart Mitochondria

EXPERIMENT 11

357

Measurement of Cholesterol and Vitamin C in

Biological Samples

371

EXPERIMENT 12

Activity and Thermal Stability of Gel-immobilized Peroxidase

EXPERIMENT 13

Extraction and Characterization of Bacterial DNA

EXPERIMENT 14

Plasmid DNA Isolation and Characterization by Electrophoresis

EXPERIMENT 15

The Action of Restriction Endonucleases on Plasmid or Viral DNA

389

399

431

Appendices

APPENDIX I

Properties of Common Acids and Bases

APPENDIX II

Properties of Common Buffer Compounds

444

APPENDIX Ml

pKa Values and pH, Values of Amino Acids

445

APPENDIX IV

Molecular Weights of Some Common Proteins

APPENDIX V

Common Abbreviations Used in This Text

APPENDIX VI

Units of Measurement

APPENDIX VII

Table of the Elements

APPENDIX VIM

Values of tfor Analysis of Statistical Confidence Limits

APPENDIX IX

Answers to Selected Study Problems INDEX

467

443

446 447

449

451

454

453

415

one

ThE O RY and Experimental

Te c h n i q_u e s

1

Introduction

to the

Biochemistry

Laboratory

Welcome to your biochemistry laboratory course! This is not the first chemistry laboratory course for most of you, but I believe you will find it to be among the most exciting and dynamic of those in which you have enrolled. Most of the experimental techniques and skills that you have acquired over the years will be of great value in this laboratory. However, you will be introduced to several new procedures and instruments. Your success in the biochemistry laboratory will depend on your mastery of these specialized techniques, use of equipment, and understanding of chemicalbiochemical principles. As you proceed through the schedule of experiments for this term, you will, no doubt, compare your work with previous laboratory experiences. In biochemistry laboratory you will seldom run reactions and isolate several hundred milligrams or a few grams of solid and liquid products as you did in organic laboratory. Rather, you will work with milligram or even microgram quantities, and in most cases the biomolecules will be extracted from plant, animal, or bacterial sources and dissolved in solution so you never really "see" the materials under study. But, you will observe the dynamic chemical and biological changes brought about by biomolecules. The techniques and procedures introduced in the laboratory will be your "eyes" and will monitor the occurrence of biochemical events.

This chapter is an introduction to procedures that are of utmost importance for the safe and successful completion of a biochemical project. It is recommended that you become familiar with the following sections before you begin laboratory work.

3

Introduction to the Biochemistry Laboratory

SAFETY IN THE LABORATORY

The concern for laboratory safety can never be overemphasized. Most students have progressed through at least two years of college laboratory work without even a minor accident. This record is, indeed, something to be proud of; however, it should not lead to overconfidence. You must always be aware that chemicals used in the laboratory are potentially toxic, irritating, or flammable. Such chemic s are a hazard, however, only when they are mishandled or improperly disposed of. It is my experience that accidents happen least often to students who come to each laboratory session mentally prepared and with a complete understanding of the experimental procedures to be followed. Since dangerous situations can develop unexpectedly, though, you must be familiar with general safety practices, facilities, and emergency action. Students must have a special concern for the safety of classmates. Carelessness on the part of one student can often cause injury to other students.

The experiments in this book are designed with an emphasis on safety. However, no amount of planning or pretesting of experiments substitutes for awareness and common sense on the part of the student. All chemicals used in the experiments outlined here must be handled with care and respect. The use of chemicals in a U.S. workplaces, including academic research and teaching laboratories, is now regulated by the Federal Hazard Communication Standard, a document written by the Occupational Safety and Health Administration (OSHA).1 Specific HA standard requires all workplaces where chemicals are used t owing: (1) develop a written hazard communication program, (2) maintain files of Material Safety Data Sheets (MSDS) on all chemicals used in that workplace, (3) abel all chemicals with information regarding hazardous properties and procedures for handling, and (4) train employees in t e proper use of these chemicals. Several states have passed "right to know" legislation which amends and expands the federal OSHA standard. If you have an interest in or concern about any chemical used in the laboratory, the MSDS for that chemical may be obtained from your instructor or laboratory manager. The actual form of an MSDS for a chemical may vary, but certain specific information must be present. Figure 1.1 is a partial copy of the MSDS for acial acetic acid, a reagent often used in bio , Different systems for labeling chemical reagent bottles are commercially available. One of the most widely used is the Hazardous Materials Identification System (HMIS). A copy of the actual label for acetic acid is shown in Figure 1.2A. The health, flammability, reactivity, and personal protection codes are defined in Figure 1.2B, It is easy to overlook some of the potential hazards of working in a biochemistry laboratory. Students often have the impression that they are working less with chemicals and more with natural biomolecules; therefore, Federal Register, Vol. 48, Nov. 25, 1983, p. 53280; Fodoral Rocjister, Vol. 50, Nov. 27, 1985, p. 48758.

A.

5

Safety in the Laboratory

Figure 1.1 Partial MSDS for glacial acetic acid. Courtesy of Sigma Chemical Co.

Section 2-Composition/Information on Ingredient Substance Name ACETIC ACID Formula

Synonyms

SARA 313 No

CAS # 64-19-7 C2H402

Acetic acid (ACGIH:OSHA), Acetic acid, glacial, Acide acetique (French), Acido acetico (Italian), Azijnzuur (Dutch), Essigsaeure (German), Ethanoic acid, Ethylic acid, Glacial acetic acid, Kyselina octova (Czech), Methanecarboxylic acid, Octowy kwas (Polish), Vinegar acid

Section 4-First Aid Measures

Oral Exposure If swallowed, wash out mouth with water provided person is conscious. Call a physician immediately. Inhalation Exposure If inhaled, remove to fresh air. If not breathing give artificial respiration. If breathing is difficult, give oxygen. Dermal Exposure In case of skin contact, flush with copious amounts of water for at least 15 minutes. Remove contaminated clothing and shoes. Call a physician. Eye Exposure In case of contact with eyes, flush with copious amounts of water for at least 15 minutes. Assure adequate flushing by separating the eyelids with fingers. Call a physician.

Section 7-Handling and Storage Handling User Exposure Do not breathe vapor. Do not get in eyes, on skin, on clothing. Avoid prolonged or repeated exposure. Storage Suitable

Keep tightly closed. Store in a cool dry place. Section 9-Physical/Chemical Appearance

Properties Col or

Form

Colorless

Molecular Weight:

60.05 AMU

Property pH

Value

BP/BP Range MP/MP Range Freezing Point

117-118°C 4°C

Vapor Pressure

11.4 mmHg 2.07 g/f

Vapor Density Saturated Vapor Cone. SC/Density

Clear liquid

At Temperature or Pressure

N/A

760 mmHg

N/A 20°C

N/A 1.06 g/cm3

Section 11-Toxicological Information Route of Exposure Skin Contact Causes burns.

Skin Absorption Harmful if absorbed through skin. Eye Contact Causes burns. Inhalation

May be harmful if inhaled. Ingestion May be harmful if swallowed. Target Organ(s) or System(s) Teeth. Kidneys. Signs and Symptoms of Exposure Material is extremely destructive to tissue of the mucous membranes and upper respiratory tract, eyes, and skin. Inhalation may result in spasm, inflammation and edema of the larynx and bronchi, chemical pneumonitis, and pulmonary edema. Symptoms of exposure may include burning sensation, coughing, wheezing, laryngitis, shortness of breath, headache, nausea, and vomiting. Ingestion or inhalation of concentrated acetic acid causes damage to tissues of the respiratory and digestive tracts. Symptoms include: hematemesis, bloody diarrhea, edema and/or perforation of the esophagus and pylorus, hematuria, anuria, uremia, albuminuria, hemolysis, convulsions, bronchitis, pulmonary edema, pneumonia, cardiovascular collapse, shock, and death. Direct contact or exposure to high concentrations of vapor with skin or eyes can cause: erythema, blisters, tissue destruction with slow healing, skin blackening, hyperkeratosis, fissures, corneal erosion, opacification, iritis, conjunctivitis, and possible blindness. To the best of our knowledge, the chemical, physical, and toxicological properties have not been thoroughly investigated.

Introduction to the Bioch
j»« -^^>^J^=J

G

below the surface of the liquid. If the tip moves above the surface of the liquid, air will be sucked into the pipet and solution will be flushed into the bulb. Other pipet fillers are used in a similar fashion. Disposable Pasteur Pipets

Often it is necessary to perform a semiquantitative transfer of a small volume (1 to 10 mL) of liquid from one vessel to another. Since pouring is not efficient, a Pasteur pipet with a small latex bulb may be used (Figure 1.4A,E). Pasteur pipets are available in two lengths (15 cm and 23 cm) and hold about 2 mL of solution. These are especially convenient for the transfer of nongraduated amounts to and from test tubes. Typical recovery while

using a Pasteur pipet is 90 to 95%. If dilution is not a problem, rinsing the original vessel with a solvent will increase the transfer yield. Used disposable pipets should be discarded in special containers for broken glass. Calibrated Pipets

Although most quantitative transfers are now done with automatic pipetting devices, which are described later in the chapter, instructions will be

E.

Quantitative Transfer of Liquids

15

Figure 1.5

How to use a Spectroline safety pipet filler. Courtesy of Spectronics Corporation, Westbury, NY 11590.

1. Using thumb and forefinger, press on valve A and squeeze bulb with other fingers to produce a vacuum for aspiration. Release valve A leaving bulb compressed.

2. Insert pipet into liquid. Press on valve S. Suction draws liquid to desired level.

3. Press on valve E to expel liquid.

4. To deliver the last drop, maintain pressure on valve E, cover E inlet with middle finger, and squeeze the small bulb.

given for the use of all types of pipets. If a quantitative transfer of a specific and accurate volume of liquid is required, some form of calibrated pipet must be used. Volumetric pipets (Figure 1.4F) are used for the delivery of liquids required in whole milliliter amounts (1, 2, 3, 4, 5, 10, 15, 20, 25, 50, and 100 mL). To use these pipets, draw liquid with a latex bulb or mechanical pipet filler to a level 2 to 3 cm above the "fill line." Touch the tip of the pipet to the inside of the glass wall of the container from which it was filled.

Introduction to the Biochemistry Laboratory

Release liquid from the pipet until the bottom of the meniscus is directly on the fill line. Transfer the pipet to the inside of the second container and release the liquid. Hold the pipet vertically, allow the solution to drain until the flow stops, and then wait an additional 5 to 10 seconds. Touch the tip of the pipet to the inside of the container to release the last drop from the outside of the tip. Remove the pipet from the container. Some liquid may still remain in the tip. Most volumetric pipets are calibrated as "TD" (to deliver), which means the intended volume is transferred withoutfinal blow-out, i.e., the pipet delivers the correct volume. Fractional volumes of liquid are transferred with graduated pipets, which are available in two types—Mohr and serological. Mohr pipets (Figure 1.4G) are available in long- or short-tip styles. Long-tip pipets are especially attractive for transfer to and from vessels with small openings. Virtually all Mohr pipets are TD and are available in many sizes (0.1 to 10.0 mL). The marked subdivisions are usually 0.01 or 0.1 mL, and the markings end a few centimeters from the tip. Selection of the proper size of pipet is especially important. For instance, do not try to transfer 0.2 mL with a 5 or 10 mL pipet. Use the smallest pipet that is practical. The use of a Mohr pipet is similar to that of a volumetric pipet. Draw the liquid into the pipet with a pipet filler to a level about 2 cm above the "0" mark. Lower the liquid level to the 0 mark. Remove the last drop from the tip by touching it to the inside of the glass container. Transfer the pipet to the receiving container and release the desired amount of solution. The solution should not be allowed to move below the last graduated mark on the pipet. Touch off the last drop. Serological pipets (Figure 1.4H) are similar to Mohr pipets, except that they are graduated downward to the very tip and are designed for blow-out. Their use is identical to that of a Mohr pipet except that the last bit of solution remaining in the tip must be forced out into the receiving container with a rubber bulb. This final blow-out should be done after 15 to 20 sec-

onds of draining. Automatic Pipetting Systems

For most quantitative transfers, including many identical small-volume transfers, a mechanical microliter pipettor (Eppendorf type) is ideal. This allows accurate, precise, and rapid dispensing of fixed volumes from 1 to 5000 jjlL (5 mL). The pipet's push-button system can be operated with one hand, and it is fitted with detachable polypropylene tips (Figure 1.6). Other useful information about pipetting is available at the Web site www.gilson.com/ pipe.htm. The advantage of polypropylene tips is that the reagent film remaining in the pipet after delivery is much less than for glass tips. Mechanical pipettors are available in up to 25 different sizes. Newer models offer continuous volume adjustment, so a single model can be used for delivery of specific volumes within a certain range. To use the pipettor, choose the proper size and place a polypropylene pipet tip firmly onto the cone as shown in Figure 1.6. Tips for pipets are

E.

17

Figure 1.6

A How to use an adjustable pipetting device. B Set the digital micrometer to the desired volume using the adjustment knob. Attach a new dis sable tip to the shaft of the pipet. Pr 's on firmly with a slight twisting motion. C Depress the plunger to the first positive stop, immerse the dis le tip into the sample liquid to a depth of 2 to 4 mm, and allow the pushbutton to r slowly to the up position and wait I to 2 seconds. D To dispense sample, place the tip end against the side wall of the re Ving vessel and depress the plunger slowly to the first stop. Wait 2 to 3 seconds, and then depress the plunger to the second stop to achieve final blow-out. Withdraw the device from the vessel carefully with the tip sliding along the inside wall of the vessel. Allow the plunger to return to the up position. Discard the tip by d ' the tip ' tor button. Photos courtesy ofRainin Instrument Company, Inc., MA. Pipetman is a registered t emark of Gilson Medical Electronics. Exclus to Rainin Instrument Company Inc.

available in several sizes, for 1-20 jjlL, 20-250 fxL5 200-1000 and 1000-5000 jjlL capacity. Details of the operation of an adjustable pipet are given in Figure 1.6. For rapid and accurate transfer of volumes greater l 5 mL, automatic repetitive dispensers are commercially available. articularly useful for the transfer of corrosive materials. The dispe which are available in several sizes, are simple to use. The vo liquid to be dispensed is mely set; the syringe plunger is li u filling and pressed downward for dispensing. Hold the receiving container under the spout while depressing the plunger. Touch off the last drop on the inside wall of the receiving container.

18

Figure 1.6 continued

OS

®ts

Special proc u

uired for cl ning I, ti up, in

er

; a

dilute

covered wi solu ' is reed o t thro

is e e tip. u

y re re

oven.

YICJ&L

su ...

If

ule or a

re

F.

Statistical Analysis of Experimental Data

19

example, if a radioactive sample is counted twice under identical experimental and instrumental conditions, the second measurement immediately following the first, the probability is very low that the numbers of counts will be identical. If the absorbance of a solution is determined several times at a

specific wavelength, the value of each measurement will surely vary from the others. Which measurements, if any, are correct? Before this question can be answered, you must understand the source and treatment of numerical variations in experimental measurements. Analysis of Experimental Data

An error in an experimental measurement is defined as a deviation of an observed value from the true value. There are two types of errors, determinate and indeterminate. Determinate errors are those that can be controlled by the experimenter and are associated with malfunctioning equipment, improperly designed experiments, and variations in experimental conditions. These are sometimes called human errors because they can be corrected or at least partially alleviated by careful design and performance of the experiment. Indeterminate errors are those that are random and cannot be con-

trolled by the experimenter. Specific examples of indeterminate errors are variations in radioactive counting and small differences in the successive measurements of glucose in a serum sample. Two statistical terms involving error analysis that are often used and misused are accuracy and precision. Precision refers to the extent of agreement among repeated measurements of an experimental value. Accuracy is defined as the difference between the experimental value and the true value for the quantity. Since the true value is seldom known, accuracy is better defined as the difference between the experimental value and the accepted true value. Several experimental measurements may be precise (that is, in close agreement with each other) without being accurate. If an infinite number of identical, quantitative measurements could be made on a biosystem, this series of numerical values would constitute a statistical population. The average of all of these numbers would be the true value of the measurement. It is obviously not possible to achieve this in practice. The alternative is to obtain a relatively small sample of data, which is a subset of the infinite population data. The significance and precision of these data are then determined by statistical analysis. This section explores the mathematical basis for the statistical treatment of experimental data. Most measurements required for the completion of the experiments can be made in duplicate, triplicate, or even quadruplicate, but it would be impractical and probably a waste of time and materials to make numerous determinations of the same measurement. Rather, when

you perform an experimental measurement in the laboratory, you will collect a small sample of data from the population of infinite values for that measurement. To illustrate, imagine that an infinite number of experimental measurements of the pH of a buffer solution are made, and the results are written on slips of paper and placed in a container. It is not feasible to

Introduction to the Biochemistry Laboratory

calculate an average value of the pH from all of these numbers, but it is possible to draw five slips of paper, record these numbers, and calculate an average pH. By doing this, you have collected a sample of data. By proper statistical manipulation of this small sample, it is possible to determine whether it is representative of the total population and the amount of confidence you should have in these numbers. The data analysis will be illustrated here primarily with the counting of radioactive materials, although it is not limited to such applications. Any replicate measurements made in the biochemistry laboratory can be analyzed by these methods. Determination of the Mean, Sample Deviation, and Standard Deviation

Radioactive decay with emission of particles is a random process. It is impossible to predict with certainty when a radioactive event will occur. Therefore, a series of measurements made on a radioactive sample will result in a series of different count rates, but they will be centered around an average or mean value of counts per minute. Table 1.1 contains such a series of count rates obtained with a scintillation counter on a single radioactive sample. A similar table could be prepared for other biochemical measurements, including the rate of an enzyme-catalyzed reaction or the protein concentration of a solution as determined by the Bradford method. The arithmetic average or mean of the numbers is calculated by totaling all the experimental values observed for a sample (the counting rates, the velocity of the reaction, or protein concentration) and dividing the total by the number of times the measurement was made. The mean is defined by Equation 1.1.

^9

x=

n

'

Equation 1.1

where

x = arithmetic average or mean

xi = the value for an individual measurement n = the total number of experimental determinations

The mean counting rate for the data in Table 1.1 is 1222. If the same radioactive sample were again counted for a series of ten observations, that series of counts would most likely be different from those listed in the table, and a different mean would be obtained. If we were able to make an infinite

number of counts on the radioactive sample, then a true mean could be calculated. The true mean would be the actual amount of radioactivity in the sample. Although it would be desirable, it is not possible experimentally to measure the true mean. Therefore, it is necessary to use the average of the

F.

21

Statistical Analysis of Experimental Data

The Observed Counts and Sample Deviation from a Typical Radioactive Sample

Counts per Minute

Sample Deviation x, - x

1243

+21

1250

+28

1201

-21

1226

+4

1220

-2

1195

-27

1206

-16

1239

+17

1220 1219

-2

-3 Mean = 1222

counts as an approximation of the true mean and to use statistical analysis to evaluate the precision of the measurements (that is, to assess the agreement among the repeated measurements). Since it is not usually practical to observe and record a measurement many times as in Table 1.1, what is needed is a means to determine the reliability of an observed measurement. This may be stated in the form of a question. How close is the result to the true value? One approach to this analysis is to calculate the sample deviation, which is defined as the difference between the value for an observation and the mean value, x (Equation 1.2). The sample deviations are also listed for each count in Table 1.1.

I

Sample deviation = xt - x

Equation 1.2

A more useful statistical term for error analysis is standard deviation, a measure of the spread of the observed values. Standard deviation, s3 for a sample of data consisting of n observations may be estimated by Equation 1.3.

s= \

2 to - *f - 1

Equation 1.3

It is a useful indicator of the probable error of a measurement. Standard deviation is often transformed to standard deviation of the mean or stan-

dard error. This is defined by Equation 1.4, where n is the number of measurements.

s

—;= Vn

Equation 1.4

22

Introduction to the Biochemistry Laboratory

Figure 1.7 The normal distribution curve.

It should be clear from this equation that as the number of experimental

observations becomes larger, sm becomes smaller, or the precision of a measurement is improved. Standard deviation may also be illustrated in graphical form (Figure 1.7). The shape of the curve in Figure 1.7 is closely approximated by the Gaussian distribution or normal distribution curve. This mathematical

treatment is based on the fact that a plot of relative frequency of a given event yields a dispersion of values centered about the mean, x. The value of xis measured at the maximum height of the curve. The normal distribution curve shown in Figure 1.7 defines the spread or dispersion of the data. The probability that an observation will fall under the curve is unity or 100%. By using an equation derived by Gauss, it can be calculated that for a single set of sample data, 68.3% of the observed values will occur within the intervals ± s3 95.5% of the observed values within x ± 2s, and 99.7% of the observed values within x ± 3s. Stated in other terms, there is a 68.3% chance

that a single observation will be in the interval x ± s. For many experiments, a single measurement is made so a mean value, x3 is not known. In these cases, error is expressed in terms of s but is defined as the percentage proportional error, %£ , in Equation 1.5. %E =

100/c

Vn

Equation 1.5

The parameter k is a proportional constant between Ex and the standard deviation. The percent proportional error may be defined within several probability ranges. Standard error refers to a confidence level of 68.3%; that is,

there is a 68.3% chance that a single measurement will not exceed the °/oEx. For standard error, k = 0.6745. Ninety-five hundredths error means there is

a 95% chance that a single measurement will not exceed the °/oEx. The constant k then becomes 1.45.

F.

23

Statistical Analysis of Experimental Data

The previous discussion of standard deviation and related statistical analysis placed emphasis on estimating the reliability or precision of experimentally observed values. However, standard deviation does not give specific information about how close an experimental mean is to the true mean. Statistical analysis may be used to estimate, within a given probability, a range within which the true value might fall. The range or confidence interval is defined by the experimental mean and the standard deviation. This simple statistical operation provides the means to determine quantitatively how close the experimentally determined mean is to the true mean. Confidence

limits (Lx and L2) are created for the sample mean as shown in Equations 1.6 and 1.7.

^9

L1 = X + (t)(sm)

Equation 1.6

Di

L2 = X - (t)(sm)

Equation 1.7

where

t = a statistical parameter that defines a distribution between a sample mean and a true mean The parameter t is calculated by integrating the distribution between percent confidence limits. Values of t are tabulated for various confidence lim-

its. Such a table is in Appendix VIII. Each column in the table refers to a desired confidence level (0.05 for 95%, 0.02 for 98%, and 0.01 for 99% confidence). The table also includes the term degrees of freedom, which is represented by n — 1, the number of experimental observations minus 1. The

values of x and sm are calculated as previously described in Equations 1.1 and 1.4.

Statistical Analysis in Practice

The equations for statistical analysis that have been introduced in this chapter are of little value if you have no understanding of their practical use, meaning, and limitations. A set of experimental data will first be presented, and then several statistical parameters will be calculated using the equations. This example will serve as a summary of the statistical formulas and will also illustrate their application.

Example 2 Ten identical protein samples were analyzed by the Bradford method for protein analysis. The following values for protein concentration were obtained.

Introduction to the Biochemistry Laboratory

Observation Number

Protein Concentration (mg/mL), x

1

1.02

2

0.98

3

0.99

4

1.01

5

1.03

6

0.97

7

1.00

8

0.98

9

1.03 1.01

10

Sample mean

-

y\x

10.02

n

10

y

Sample deviation

Sample deviation = xl - x Observation

x,-x

1

+0.02

2

-0.02 -0.01 +0.01 +0.03 -0.03

3 4

5

6 7

8

9 10

0.00 -0.02 +0.03 +0.01

Calculation of the sample deviation for each measurement gives an indication of the precision of the determinations. Standard deviation

s = 0.02

The mean can now be expressed as x ± s (for this specific example, 1.00 ± 0.02 mg/mL). The probability of a single measurement falling within these limits is 68.3%. For 95.5% confidence (2s), the limits would be 1.00 ± 0.04 mg/mL.

Study Problems

25

Standard error of the mean

_

s

Sm~v?i _ 0.02

s™"VTo sm = 0.006

This statistical parameter can be used to gauge the precision of the experimental data. Confidence limits

The desired confidence limits will be set at the 95% confidence level. There-

fore we will choose a value for t from Appendix VIII in the column labeled

'0.05 and » - 1 = 9. L1=^+(f0.05)(Sm)

L2 = 3?-(f0.05)(Sm) sm = 0.006 fa05 = 2.262 x = 1.00

L1 = 1.00+ (2.262)(0.006) L1 = 1.01

L2 = 0.99 We can be 95% confident that the true mean falls between 0.99 and 1.01

mg/mL. Study Problems

1. Define each of the following terms. (a) OSHA (e) Purified water (b) MSDS (f) Error

26

Introduction to the Biochemistry Laboratory

B

(c) Flowchart (g) Standard deviation (d) Pasteur pipet (h) Molarity 2. What personal protection items must be worn when handling glacial acetic acid?

B

B

3. Draw a schematic picture of your biochemistry lab and mark locations of the following safety features: eyewashes, first-aid kit, shower, fire extinguisher, chemical spill kits, and direction to nearest exit. 4. Describe how you would prepare a 1-liter aqueous solution of each of the following reagents: (a) 1 M glycine (b) 0.5 M glucose (c) 10 mM ethanol (d) 100 nM hemoglobin 5. Describe how you would prepare just 10 mL of each of the solutions in Problem 4.

B

6. If you mix 1 mL of the 1 M glycine solution in Problem 4 with 9 mL of water, what is the final concentration of this diluted solution in mM?

B

Z Convert each of the concentrations below to mM and jjlM. (a) 10 mg of glucose per 100 mL (b) 100 mL of a solution 2% in alanine

B

8. You have just prepared a solution by weighing 20 g of sucrose, transferring it to a 1-liter volumetric flask, and adding water to the line. Calculate the concentration of the sucrose solution in terms of mM, mg/mL, and % (wt/vol).

B

9. The concentrations of cholesterol, glucose, and urea in blood from a fasting individual are listed below in units of mg/100 mL (sometimes called mg°/o). These are standard concentration units used in the clinical chemistry lab. Convert the concentrations to mM. cholesterol—200 mg°/o glucose—75 mg% urea-20 mg°/o

B 10. The following optical rotation readings were taken by a polarimeter on a solution of an unknown carbohydrate. (a) Calculate the sample mean. (b) Calculate the standard deviation. (c) Calculate the 95% confidence levels for the measurement. aobs (degrees) +3.24 +3.15 +3.30

+3.20 +3.21 +3.19

+3.17 +3.23 +3.20

+3.25

Further Reading

27

Further Reading Data Analysis

P. Meir and R. Zund, Statistical Methods in Analytical Chemistry (1993), John Wiley & Sons (New York). J. Miller and J. Miller, Statistics for Analytical Chemistry, 3rd ed. (1992), PrenticeHall (Englewood Cliffs, NJ). J. Robyt and B. White, Biochemical Techniques: Theory and Practice (1987), Waveland Press (Prospect Heights, IL). Reagents and Solutions

M. Brush, The Scientist, June 8, 1998, pp. 18-23. A discussion of water purification. S. Kegley and J. Andrews, The Chemistry of Water (1997), University Science Books (Sausalito, CA). A discussion of water purity and analysis.

J. Risley,/ Chem. Educ. 68,1054-1055 (1991). "Preparing Solutions in the Biochemistry Lab." Safety

M. Armour, Hazardous Laboratory Chemicals Disposal Guide, 2nd ed. (1996), CRC Press (Boca Raton, FL).

K. Barker, Editor, At the Bench: A Laboratory Navigator (1998), Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY).

C. Gorman, Editor, Working Safely with Chemicals in the Laboratory, 2nd ed. (1994), Genium Publishing Corp. (Schenectady, NY). G. Lowry and R. Lowry, Lowry's Handbook ofRight-to-Know and Emergency Planning (1990), Lewis Publishers (Chelsea, MI). Prudent Practices in the Laboratory: Handling and Disposal of Chemicals (1995), National Research Council, National Academy Press (Washington, DC).

Safety in Academic Chemical Laboratories, 6th ed. (1995), American Chemical Society (Washington, DC). J. Young, Editor, Improving Safety in the Chemical Laboratory—A Practical Guide, 2nd ed. (1991), Wiley-Interscience (New York). Writing Laboratory Reports

K. Barker, Editor, At the Bench: A Laboratory Navigator (1998), Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY). Covers several important topics including lab orientation, keeping a notebook, and lab procedures. H. Beall and J. Trimber, A Short Guide to Writing about Chemistry (1996), BenjaminCummings (Menlo Park, CA). R. Day, How to Write and Publish a Scientific Paper, 2nd ed. (1988), Oryx Press (Phoenix).

Introduction to the Biochemistry Laboratory

R. Dodd, Editor, The ACS Style Guide: A Manualfor Authors and Editors, 2nd ed. (1997), American Chemical Society (Washington, DC). H. Ebel, C. Bliefert, and W Russey, The Art of Scientific Writing: From Student Reports to Professional Publications in Chemistry and Related Fields (1997), John Wiley & Sons (New York). C. Lobban and M. Schefter, Successful Lab Reports: A Manualfor Science Students (1992), Cambridge University Press (Cambridge).

J. Walker, Biochem. Educ. 19,31-32 (1991). "A Student's Guide to Practical Write-ups." On the Web

http://www.graphpad.com/prism/Prism.htm Software for statistics and curve fitting. http://www.statistics.com/ Click on Free Web-based software for data analysis.

http://www.gilson.com/pipe.htm Information on automatic pipets, procedures for use, and helpful hints. http://ehs.clemson.edu/bsm-spil.html Biological Safety Manual. http://www.hendrix.edu/chemistry/chemsafe.htm Information on chemical hygiene and safety with links to MSDS searches. http://research.nwfsc.noaa.gov/msds.html Links to MSDS searches.

http://www.osha.gov/ Review of functions and regulatory procedures by OSHA. http://www.dixie.edu/mort/manual/mechanics/Notebook.html. How and why to keep a notebook. Procedures for use and helpful hints. http://practicingsafescience.org Advice from the Howard Hughes Medical Institute.

2

General

Laboratory

Procedures

All biochemical laboratory activities, whether in education, research, or industry, are replete with techniques that must be carried out almost on a daily basis. This chapter outlines the theoretical and practical aspects of some of these general and routine procedures, including use of buffers, pH and other electrodes, dialysis, membrane filtration, lyophilization, centrifugal concentration, and quantitative methods for protein and nucleic acid measurement.

A.

PH, BUFFERS, ELECTRODES, AND BIOSENSORS

Most biological processes in the cell take place in a water-based environment. Water is an amphoteric substance; that is, it may serve as a proton donor (acid) or a proton acceptor (base). Equation 2.1 shows the ionic equilibrium of water.

^3

H20 ^^ H+ + OH-

Equation 2.1

In pure water, [H+] = [OH-] = 10-7; in other words, the pH or -log [H+] is 7. Acidic and basic molecules, when dissolved in water in a biological cell or test tube, react with either H+ or OH~ to shift the equilibrium of Equation 2.1 and result in a pH change of the solution. Biochemical processes occurring in cells and tissues depend on strict regulation of the hydrogen ion concentration. Biological pH is maintained at a constant value by natural buffers. When biological processes are studied in vitro, artificial media must be prepared that mimic the cell's natural 29

General Laboratory Procedures

environment. Because of the dependence of biochemical reactions on pH, the accurate determination of hydrogen ion concentration has always been of major interest. Today, we consider the measurement and control of pH to be a simple and rather mundane activity. However, an inaccurate pH measurement or a poor choice of buffer can lead to failure in the biochemistry laboratory. You should become familiar with several aspects of pH measurement, electrodes, and buffers. Measurement of pH

A pH measurement is usually taken by immersing a glass combination electrode into a solution and reading the pH directly from a meter. At one time, pH measurements required two electrodes, a pH-dependent glass electrode sensitive to H+ ions and a pH-independent calomel reference electrode. The potential difference that develops between the two electrodes is measured as a voltage as defined by Equation 2.2. HI

"i

,,

,_

V= Constant +

2.303R7 ,,

j.

PH

Equation 2.2

where ta int _

R =

T= F=

voltage of the completed circuit potential of reference electrode the gas constant the absolute temperature the Faraday constant

A pH meter is standardized with buffer solutions of known pH before a measurement of an unknown solution is taken. It should be noted from

Equation 2.2 that the voltage depends on temperature. Hence, pH meters must have some means for temperature correction. Older instruments usually have a knob labeled "temperature control," which is adjusted by the user to the temperature of the measured solution. Newer pH meters automatically display a temperature-corrected pH value. Most pH measurements today are obtained using a single combination electrode (Figure 2.1). Both the reference and the pH-dependent electrode are contained in a single glass or plastic tube. Although these are more expensive than dual electrodes, they are much more convenient to use, especially for smaller volumes of solution. Using a pH meter with a combination electrode is relatively easy, but certain guidelines must be followed. A pH meter not in use is left in a "standby" position. Before use, check the level of saturated KC1 in the electrode. If it is low, check with your instructor for the filling procedure. Turn the temperature control, if available, to the temperature of the standard calibration buffers and the test solutions. Be sure the

function dial is set to pH. Lift the electrode out of the storage solution, rinse it with distilled water from a wash bottle, and gently clean and dry the elec-

A.

pH, Buffers, Electrodes, and Biose

31

>rs

me 2J

e combination pH e. Courtesy of Hanna ents.

Electrodes are housed in either

plastic or an all-glass body configun. They can be either single or as shown in the diagram, bined into one body for ease of . Regardless of the configura, there are several features cornodes.

brane glass: actual measurement.

n: Acts as a liquid

i

. Inte con

r

I conductor.

: Supplies a brium voltage.

. pH ent: Supplies a voltage based on the pH value of the sample. . Reference fill hole: Used to

replace the reference electrolyte solution. Plastic

Glass

body

body

trode with a tissue. Immerse the electrode in a standard buffer. Common

standard buffers are pH 4, 7, and 10 with accuracy of ±0.02 pH unit. The standard buffer should have a p ' hin two pH units of the expected pH of the test solution. The bulb oft electrode must be completely covered with solution. Turn the pH meter to "on" or "read" and adjust the meter with the "calibration dial" (sometimes called "intercept") until the proper pH of the standard buffer is indicated on the dial. Turn the pH meter to standby position. Remove the electrode d again rinse with distilled water and carefully blot dry with tissue. Immerse the electrode in a standard buffer of different pH and turn the p eter to "read." The dial should read within ±0.05 pH unit of the kno alue. If it does not, adjust to the proper pH and again check the first s ard pH buffer. Clean the electrode and immerse it in the test solution. Record the pH of the test solution. As with all delicate equipment, the pH meter and electrode must receive proper care and maintenance. All electrodes should be kept clean and stored in solutions suggested by manufacturers. Glass electrodes are fragile and expensive, so they must be handled with care. If pH measurements of protein solutions are often taken, a protein film may develop on the electrode; it can be removed by soaking in 5% pepsin in 0.1 M HC1 for 2 hours and rinsing well with water.

General Laboratory Procedures

Measurements of pH are always susceptib Some common problems are:

to experimental errors.

1. The Sodium Error Most glass combination electrodes are sensitive to Na+ as well as H+. The sodium error can become qui " nificant at high pH values, where 0.1 M Na+ may decrease the measur H by 0.4 to 0.5 unit. Several things may be done to reduce the sodium error. Some commercial suppliers of e odes provide a standard curve for sodium error correction. Newer ele des that are virtual Na+ impermeable are now commercia

availabl

electrode is vailable,

neither a standard curve nor a sodium-insensitive

ssium salts may be substituted for sodium salts.

2. Con titration Effects The pH of a solution varies with the concentration of buffer ions or other salts in the s tion. This is because the pH of a solution depends on the activity of an i ic species, not on the concentration. Activity, you may recall, is a ther dynamic term used to define species in a nonideal so ution. At infinite d tion, the activity of a species is equivalent to its con ntration. At finite lutions, however, the activity of a solute and its cone tration are not eq It is common prac ce in biochemical oratories to prepare concentrated "stock" solution and buffers. These are then diluted to the proper concentration when ne

ed. Because of the concentration effects described

above, it is important t adjust the pH of these solutions after dilution. 3. Temperature Effec e pH of a buffer solution is influenced by temperature. This effec is to a temperature-dependent change of the

dissociation constant (

a

ions in solution. The pH of the commonly

used buffer Tris is great a ed by temperature changes, with a ApK /C° of —0.031. This means that 7.0 Tris buffer made up at 4°C would ave a pH of 5.95 at 37°C. The way to avoid this problem is to prepare the buffer solution at the temperature at w ich it will be used and to standardize the electrode with buffers at the same temperature as the solution you wish to measure. Biochemical Buffers

Buffer ions are used to maintain solutions at constant pH values. e selection of a buffer for use in the investigation of a biochemical process is of critical importance. Before the characteristics of a buffer system are discussed, we will review some concep in acid-base chemistry. Weak acids and bases do not completely dissociate in solution but exist as equilibrium mixtures (Equation 2.3). HA ^=i

H' + A

Equation 2.3

k2

HA represents a weak acid and A" represents its conjugate base; kx represents the rate constant for dissociation of he acid and k2 the rate constant

A.

pH, Buffers, Electrodes, and Biosensors

33

for association of the conjugate base and hydrogen ion. The equilibrium

constant, Ka, for the weak acid HA is defined by Equation 2.4. „

k,

[H+][A-]

K* = *TW

Equa"on2-4

which can be rearranged to define [H+] (Equation 2.5).

| [H+]=M^

Equation 2.5

The [H+] is often reported as pH, which is —log [H+]. In a similar fashion,

-log Ka is represented by pKa. Equation 2.5 can be converted to the -log form by substituting pH and pKa: fA~l

pH = pKa + log ——

Equation 2.6

Equation 2.6 is the familiar Henderson-Hasselbalch equation, which defines the relationship between pH and the ratio of acid and conjugate base concentrations. The Henderson-Hasselbalch equation is of great value in buffer chemistry because it can be used to calculate the pH of a solution if the mo-

lar ratio of buffer ions ([A-]/[HA]) and the pKa of HA are known. Also, the molar ratio of HA to A~ that is necessary to prepare a buffer solution at a

specific pH can be calculated if the pKa is known. A solution containing both HA and A~ has the capacity to resist changes in pH; i.e., it acts as a buffer. If acid (H+) were added to the buffer solution, it would be neutralized by A~ in solution: H+ + A- —» HA

Equation 2.7

Base (OH-) added to the buffer solution would be neutralized by reaction with HA:

I

OH- + HA —> A- + H20

Equation 2.8

The most effective buffering system contains equal concentrations of the acid, HA, and its conjugate base, A~. According to the Henderson-

Hasselbalch equation (2.6), when [A~] is equal to [HA], pH equals pKa. Therefore, the pKa of a weak acid-base system represents the center of the buffering region. The effective range of a buffer system is generally two pH

units, centered at the pKa value (Equation 2.9).

I

Effective pH range for a buffer = pKa ± 1

Equation 2.9

34

General Laboratory Procedur

Selection of a Biochemical Buffer

Virtually all biochemical investigations must be carried out in buffered aqueous solutions. The natural environment of biomolecu nd cellular organelles is under strict pH control. When these compone e extracted from cells, they are most stable if maintained in their normal range, usually 6 to 8. An artificial buffer system is found to be the best substitute for the natural cell milieu. It should also be recognized that many biochemical processes (especially some enzyme processes) produce or consume hydrogen ions. The buffer system neutralizes these solutions and maintains a constant chemical environment.

Although most biochemical solutions require buffer systems effective in the p range 6 to 8, there is occasionally a need for buffering over the pH range 2 to 12. Obviously, no single acid-conjugate base pair will be effective over this entire range, but several buffer systems are availab that may be used in discrete pH ranges. Figure 2.2 compares the effective buffering ranges of common biological buffers. It should be noted that some

buffers (phosphate, succinate, and citrate) have more than one pKa value, so they may be used in different pH regions. Many buffer systems are effec-

Figure 2.2

Effective buffering ranges

HEPES

of several common buffers. Histidine

Bis-Tris T"

Bicine

MES

CAPS

imidazole

Tris

Formate

—i—

Ace

te

Succinate

Citrate

Citrate

!

i

Phosphate

te

10

PH

11

12

A.

pH, Buffers, Electrodes, and Biosensors

35

tive in the usual biological pH range (6 to 8); however, there may be major problems in their use. Several characteristics of a buffer must be considered before a final selection is made. The molecular weights and pK values of several common buffer compounds are listed in Appendix II. Following is a discussion of the advantages and disadvantages of the commonly used buffers.

Phosphate Buffers

The phosphates are among the most widely used buffers. These solutions have high buffering capacity and are very useful in the pH range 6.5 to 7.5. Because phosphate is a natural constituent of cells and biological fluids, its presence affords a more "natural" environment than many buffers. Sodium or potassium phosphate solutions of all concentrations are easy to prepare using the Henderson-Hasselbalch equation. The major disadvantages of phosphate solutions are (1) precipitation or binding of common biological cations (Ca2+ and Mg2+), (2) inhibition of some biological processes, including some enzymes, and (3) limited useful pH range. Zwitterionic Buffers (Good's Buffers)

In the mid-1960s, N. E. Good and his colleagues recognized the need for a set of buffers specifically designed for biochemical studies (Good and Izawa, 1972). He and others noted major disadvantages of the established buffer systems. Good outlined several characteristics essential in a biological buffer system:

1.

pKa between 6 and 8.

2. 3.

Highly soluble in aqueous systems. Exclusion or minimal transport by biological membranes.

4.

Minimal salt effects.

5.

Minimal effects on dissociation due to ionic composition, concentration, and temperature. Buffer-metal ion complexes nonexistent or soluble and well defined. Chemically stable. Insignificant light absorption in the ultraviolet and visible regions. Readily available in purified form.

6. 7. 8. 9.

Good investigated a large number of synthetic zwitterionic buffers and found many of them to meet these criteria. Table 2.1 lists several of these buffers and their properties. Good's buffers are widely used, but their main disadvantage is high cost. Some Good's buffers, such as Tris, HEPES, and PIPES, have been shown to produce radicals under a variety of experimental conditions, so they should be avoided if biological redox processes or radical-based reactions are being studied. Radicals are not produced from MES or MOPS buffers.

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biological response

Equation 3.9

In a biological system, the formation of the complex often triggers some response such as immunological action, control of a metabolic process, hormone action, catalytic breakdown of a substrate, or membrane transport. The biological response depends on proper molecular recognition and binding as shown in the reaction. The most common example of Equation 3.9 is the interaction that occurs between an enzyme molecule, E, and a substrate, S, with reversible formation of an ES complex. The biological event resulting from this interaction is the transformation of S to a metabolic product, P. Only the first step in Equation 3.9, formation of the complex, is of concern in affinity chromatography. In practice, affinity chromatography requires the preparation of an insoluble stationary phase, to which appropriate ligand molecules (B) are covalently affixed. Thus, ligand molecules are immobilized on the stationary support. The affinity support is packed into a column through which a mixture containing the desired macromolecule, A, is allowed to percolate. There are many types of molecules in the mixture, especially if it is a crude cell extract, but only macromolecules that recognize and bind to immobilized B are retarded in their movement through the column. After the nonbinding molecules have washed through the column, the desired macromolecules, B,

100

Purification and Identification of Biomolecules by Chromatography

Figure 3.18

The steps of affinity

chromatography.

_

1 • Attach ''9and B to 9el:

Gel

Modified gel

2. Pack modified of components

B 3. Dissociate com

into column and adsorb sample containing a mixture , C, and D:

A, C,D

(

K~—B:A + C, D

x with Y and elute A;

■B:A + Y

C^)

B:Y + A

are eluted by gentle disruption of the A:B complex. Study Figure 3.18 for an illustration of affinity chromatogr Affinity chromatography can plied to the isolation and purification of virtually all biological macromolecules. It has been used to purify nucleic acids, enzymes, transport proteins, antibodies, hormone receptor proteins, drug-binding proteins, neurotransmitter proteins, and many others. Successful application of affinity chromatography requires careful design of experimental conditions. The essential components, which are outlined below, are (1) creation and preparation of a stationary matrix with immobilized ligand and (2) design of column development and eluting conditions.

Chromatographic Media

Selection of the matrix used to immobilize a ligand requires consideration of several properties. The stationary supports used in gel exclusion chromatography are found to be quite suitable for affinity chromatography because (1) they are physically and chemically stable under most experimental conditions, (2) they are relatively free of nonspecific adsorption effects, (3) they have satisfactory flow characteristics, (4) they are available with very large pore sizes, and (5) they have reactive functional groups to which an appropriate ligand may be attached. Four types of media possess most of these desirable characteristics: agarose, polyvinyl, polyac amide, and controlled-porosity glass (CPG) bea s. Highly porous agarose beads such as Sepharose 4B (Pharmacia) and Bio-Gel A-150 m (Bio-Rad Laboratories) have virtually all of u e characteristics and are the most widely used matrices. Polyacrylamid s such as Bio-Gel P-300 (Bio-Rad) display many of the recommended "es; however, the porosity is not esx igh.

H.

Affinity Chromatography and Immunoadsorption

101

The Immobilized Ligand

The ligand (B in Equation 3.9 and Figure 3.18) can be selected only after the nature of the macromolecule to be isolated is known. When a hormone re-

ceptor protein is to be purified by affinity chromatography, the hormone itself is an ideal candidate for the ligand. For antibody isolation, an antigen or hapten may be used as ligand. If an enzyme is to be purified, a substrate analog, inhibitor, cofactor, or effector may be used as the immobilized ligand. The actual substrate molecule may be used as a ligand, but only if column conditions can be modified to avoid catalytic transformation of the bound substrate.

In addition to the foregoing requirements, the ligand must display a strong, specific, but reversible interaction with the desired macromolecule and it must have a reactive functional group for attachment to the matrix. It should be recognized that several types of ligand may be used for affinity purification of a particular macromolecule. Of course, some ligands will work better than others, and empirical binding studies can be performed to select an effective ligand. Attachment of Ligand to Matrix

Several procedures have been developed for the covalent attachment of the ligand to the stationary support. All procedures for gel modification proceed in two separate chemical steps: (1) activation of the functional groups on the matrix and (2) joining of the ligand to the functional group on the matrix.

A wide variety of activated gels is now commercially available. The most widely used are described as follows: Cyanogen Bromide-Activated Agarose

This gel is especially versatile because all ligands containing primary amino groups are easily attached to the agarose. It is available under the trade name CNBr-activated Sepahrose 4B (Pharmacia). Since the gel is extremely reactive, very gentle conditions may be used to couple the ligand. One disadvantage of CNBr activation is that small ligands are coupled very closely to the matrix surface; macromolecules, because of steric repulsion, may not be able to interact fully with the ligand. The procedure for CNBr activation and ligand coupling is outlined in Figure 3.19A. 6-Aminohexanoic Acid (CH)-Agarose and 1,6-Diaminohexane (AH)-Agarose

These activated gels overcome the steric interference problems stated above by positioning a six-carbon spacer arm between the ligand and the matrix. Ligands with free primary amino groups can be covalently attached to CH-agarose, whereas ligands with free carboxyl groups can be coupled to

102

Purification and Identification of Biomolecules by Chromatography CHAPTER 3

AH-agarose. The attachment of ligands to AH and CH gels is outlined in Figure 3.19B,C. Carbonyldiimidazole (CDI)-Activated Supports

Reaction with CDI produces gels that contain uncharged 7V-alkylcarbamate groups (see Figure 3.19D). CDI-activated agarose, dextran, and polyvinyl acetate are sold by Pierce Chemical Co. under the trade name Reacti-Gel. Epoxy-Activated Agarose

The structure of this gel is shown in Figure 3.19E. It provides for the attachment of ligands containing hydroxyl, thiol, or amino groups. The hydroxyl groups of mono-, oligo-, and polysaccharides can readily be attached to the gel. Epoxy-activated Sepharose 6B is available from Pharmacia. Group-Specific Adsorbents

The affinity materials described up to this point are modified with a ligand having specificity for a particular macromolecule. Therefore, each time a biomolecule is to be isolated by affinity chromatography, a new adsorbent Figure 3.19

Attachment of specific ligands to activated gels. R = ligand.

A CNBR-agarose

gelv

+ CNBr-

gel

X)H

^CL\

/

^Cr

C = NH

RNhL

Vgel

/OH

/

^O—C—NHR + NH0

B AH-agarose O

gel— NH — (CH2)6— NH2 + RCOOH

gel — NH— (CH2)6— NHC— R

c CH-agarose

q

gel— NH — (CH2)5COOH + RNH2

gel — NH — (CH2)5— C — NHR

D Carbonyldiimidazole-agarose O

II

O

/^N

gel—O —C —N

J + RNH2

||

gel —O —C —NHR

E Epoxy-activated agarose

gel — O — CH2— CH — CH2 + ROH

O

gel — O — CH2— CH — CH2

I

OH

I

O—R

H.

Affinity Chromatography and Immunoadsorption

10 1

Group-Specific Adsorbents Useful in Biochemical Applications

Group-Specific Adsorbent 5'-AMP-agarose

Group Specificity Enzymes that have NAD+ cofactor; ATP-dependonl kinases

Benzamidine-Sepharose

Serine proteases

Boronic acid-agarose

Compounds with c/s-diol groups; sugars, catecholamines, ribonucleotides, glycoproteins

Cibracron blue-agarose

Enzymes with nucleotide cofactors (dehydrogenases, kinases, DNA polymerases); serum albumin

Concanavalin A-agarose

Glycoproteins and glycolipids

Heparin-Sepharose

Nucleic acid-binding proteins, restriction endonucleases, lipoproteins

Iminodiacetate-agarose

Proteins with affinity for metal ions, serum proteins, interferons

Lentil lectin-Sepharose

Detergent-soluble membrane proteins

Lysine-Sepharose

Nucleic acids

Octyl-Sepharose

Weakly hydrophobic proteins, membrane proteins

Phenyl-Sepharose

Strongly hydrophobic proteins

Poly(A)-agarose

Nucleic acids containing poly(U) sequences, mRNA-binding proteins

Poly(U)-agarose

Nucleic acids containing poly(A) sequences, poly(U)-binding proteins

Protein A-agarose

IgG-type antibodies

Thiopropyl-Sepharose

— SH containing proteins

must be designed and prepared. Ligands of this type are called substance specific. In contrast, group-specific adsorbents contain ligands that have affinity for a class of biochemically related substances. Table 3.6 shows several commercially available group-specific adsorbents and their specificities. The principles behind binding of nucleic acids and proteins to group-specific adsorbents depend on the actual affinity adsorbent. In most cases, the immobilized ligand and macromolecule (protein or nucleic acid) interact through one or more of the following forces: hydrogen bonding, hydrophobic interactions, and/or covalent interactions. Some group-specific adsorbents deserve special attention. Phenyl- and octyl-Sepharose are gels used for hydrophobic interaction chromatography. These adsorbent materials separate proteins on the basis of their hydrophobic character. Because most proteins contain hydrophobic amino acid side chains, this method is widely used. OctylSepharose is strongly hydrophobic; hence it binds strongly to nonpolar proteins. Phenyl-Sepharose is more weakly hydrophobic; therefore, it is more likely to reversibly bind strongly hydrophobic proteins. The use of thiopropyl-Sepharose and boronic acid-agarose is an example of covalent chromatography, since relatively strong but reversible covalent bonds are formed between the affinity gel and specific macromolecules.

Purification and Identification of Biomolecules by Chromatography

Metal affinity chromatography is a relatively new method that separates proteins on the basis of metal binding. This technique is used in Experiment 4 to isolate a-lactalbumin from milk.

The availability of a great variety of group-specific adsorbents in prepacked columns makes possible the combination of FPLC and affinity chromatography for the separation and purification of proteins. One of the most specific modifications of affinity chromatography is immunoaffinity. The unique high specificity of antibodies for their antigens is valuable for the purification of antigens. In practice, the antibody is immobilized on a column support. When a mixture containing several other proteins along with the protein antigen for the antibody is passed through the column, only the antigen binds; the other proteins, which have no affinity for the antibody, wash off the column. Protein A-agarose in Table 3.6 is an example of immunoaffinity; however, this adsorbent does not recognize specific antibodies but, rather, the general family of immunoglobulin G antibodies.

Experimental Procedure for Affinity Chromatography

Although the procedure is different for each type of substance isolated, a general experimental plan is outlined here. Figure 3.20 provides a stepby-step plan in flowchart form. Many types of matrix-ligand systems are commercially available and the costs are reasonable, so it is not always necessary to spend valuable laboratory time for affinity gel preparation. Even if a specific gel is not available, time can be saved by purchasing preactivated gels for direct attachment of the desired ligand. Once the gel is prepared, the procedure is similar to that described earlier. The major difference is the use of shorter columns. Most affinity gels have high capacities and column beds less than 10 cm in length. A second difference is the mode of elution. Ligand-macromolecule complexes immobilized on the column are held together by hydrogen bonding, ionic interactions, and hydrophobic effects. Any agent that diminishes these forces causes the release and elution of the macromolecule from the column. The common meth-

ods of elution are change of buffer pH, increase of buffer ionic strength, affinity elution, and chaotropic agents. The choice of elution method depends on many factors, including the types of forces responsible for complex formation and the stability of the ligand matrix and isolated macromolecule.

Buffer pH or Ionic Strength

If ionic interactions are important for complex formation, a change in pH or ionic strength weakens the interaction by altering the extent of ionization of ligand and macromolecule. In practice, either a decrease in pH or a gradual increase in ionic strength (continual or stepwise gradient) is used.

H.

105

Affinity Chromatography and Immunoadsorption

Figure 3.20

Is matrix-ligand available?

Experimental procedure for affinity chromatography.

NO

YES

Select gel and ligand

Swell gel in water or buffer

Couple ligand

Prepare gel for column

Pack gel in glass column and set up column equipment

Equilibrate column with buffer

Apply sample

Wash column to remove unbound molecules

Elute bound molecules

Collect and analyze eluent

Regenerate and store gel

Affinity Elution

In this method of elution, a selective substance added to the eluting

buffer competes for binding to the ligand or for binding to the adsorbed macromolecule. Chaotropic Agents

If gentle and selective elution methods do not release the bound macromolecule, then mild denaturing agents can be added to the buffer. These

106

Purification and Identification of Biomolecules by Chromatography CHAPTER 3

substances deform protein and nucleic acid structure and decrease the stability of the complex formed on the affinity gel. The most useful agents are

urea, guanidine • HO, CNS", CIO", and CCl3COO". These substances

^N 0

5

10

15

20

25

Elution volume (ml_) Figure 3.21

Purification of a-chymotrypsin by affinity chromatography on immobilized D-tryptophan methyl ester. From Affinity Chromatography: Principles and Methods, Pharmacia, Uppsala, Sweden.

should be used with care, because they may cause irreversible structural changes in the isolated macromolecule. The application of affinity chromatography is limited only by the imagination of the investigator. Every year literally hundreds of research papers appear with new and creative applications of affinity chromatography. Figure 3.21 illustrates the purification of a-chymotrypsin by affinity chromatography on immobilized D-tryptophan methyl ester, a-Chymotrypsin can recognize and bind, but not chemically transform, D-tryptophan methyl ester. The enzyme catalyzes the hydrolysis of L-tryptophan methyl ester. The impure a-chymotrypsin mixture was applied to the gel, D-tryptophan methyl ester coupled to CH-Sepharose 4B, and the column washed with Tris buffer. At the point shown by the arrow, the eluent was changed to 0.1 M acetic acid. The decrease in pH caused release of a-chymotrypsin from the column.

PERFUSION CHROMATOGRAPHY

A separation method that improves resolution and decreases the time required for analysis of biomolecules has recently been introduced. This method, called perfusion chromatography, relies on a type of particle support called POROS, which may be used in low-pressure and high-pressure liquid chromatography applications. In conventional chromatographic separations, some biomolecules in the sample move rapidly around and past the media particles while other molecules diffuse slowly through the particles (Figure 3.22A). The result is loss of resolution because some biomolecules exit the column before others. To improve resolution, the researcher with conventional media found it necessary to reduce the flow rate to allow for diffusion processes, increasing the time required for analysis. In other words, before the development of perfusion chromatography, the researcher had to choose between high speed-low resolution and low speed-high resolution. POROS particles have two types of pores-through pores (60008000 A in diameter), which provide channels through the particles, and connected diffusion pores (800-1500 A in diameter), which line the through pores and have very short diffusion path lengths (Figure 3.22B). This combination pore system increases the porosity and the effective surface area of the particles and results in improved resolution and shorter analysis times (30 seconds to 3 minutes for POROS versus 30 minutes to several hours for conventional media). POROS media, made by copolymerization of styrene and divinylbenzene, have high mechanical strength and are resistant to many solvents and chemicals. The functional surface chemistry of the particles can be modified to provide supports for many types of chromatography, including ion exchange, hydrophobic interaction, immobilized metal affinity, reversed

I.

107

Perfusion Chromatography

Figure 3.22

Transport of biomolecules through chromatographic media. A Conventional support particles. B POROS particles for perfusion chromatography. Courtesy of PerSeptive Biosystems, Cambridge, MA.

phase, group-selective affinity, and conventional bioaffinity. Perfusion chromatography has been applied with success to the separation of peptides, proteins, and polynucleotides on both preparative and analytical scales. In addition to high resolution and short analysis times, perfusion chromatography has the advantage of improved recovery of biological activity because active biomolecules spend less time on the column, where denaturing conditions may exist. Study Problems

1. Amino acid analyzers are instruments that automatically separate amino acids by cation-exchange chromatography Predict the order of elution (first to last) for each of the following sets of amino acids at pH = 4. (a) Gly, Asp, His (b) Arg, Glu, Ala (c) Phe, His, Glu

2. Predict the relative order of paper chromatography R{ values for the amino acids in the following mixture: Ser, Lys, Leu, Val, and Ala. Assume that the developing solvent is w-butanol, water, and acetic acid. 3. In what order would the following proteins be eluted from a DEAE-

cellulose ion exchanger by an increasing salt gradient. The pHt is listed for each protein. Egg albumin, 4.6 Pepsin, 1.0 Serum albumin, 4.9

Cytochrome c, 10.6 Myoglobin, 6.8 Hemoglobin, 6.8

4. Draw the elution curve {A2m vs. fraction number) obtained by passing a mixture of the following proteins through a column of Sephadex G-100. The molecular weight is given for each protein.

Purification and Identification of Biomolecules by Chromatography

Myoglobin, 16,900

Myosin, 524,000

Catalase, 222,000

Serum albumin, 68,500

Cytochrome c, 13,370

Chymotrypsinogen, 23,240



5. Amino acids and fatty acids do not readily elute from gas chromatography columns even at temperatures above 200°C. What can be done to these biomolecules to allow gas chromatographic analysis?



6. Describe the various detection methods that can be used in HPLC.



7. 8.



9.

10.

What types of biomolecules are detected by each method? Name three enzymes that you predict will bind to the affinity support, 5'-AMP-agarose. Briefly describe how you would experimentally measure the exclusion limit for a Sephadex gel whose bottle has lost its label. Describe how you would use "affinity elution" to remove the enzyme alcohol dehydrogenase bound to a Cibracron blue-agarose column. Explain the elution order of amino acids in Figure 3.13.

Further Reading

H. Barth and B. Boyes, Anal Chem. 62, 382R-394R (1990). "Size Exclusion Chromatography."

R. Boyer, Concepts in Biochemistry (1999), Brooks/Cole (Pacific Grove, CA), pp. 102-104. An introduction to chromatography. M. Chakravarthy, L. Snyder, T. Vanyo, J. Holbrook, and H. Jakubowski,/. Chem. Educ. 73,268-272 (1996). Protein structure and chromatographic behavior. S. Fulton and D. Vanderburgh, The Busy Researcher's Guide to Biomolecule Chromatography (1996), PerSeptive Biosystems (Framingham, MA). Practical guide to chromatography of biomolecules with emphasis on perfusion methods.

R. Garrett and C. Grisham, Biochemistry, 2nd ed. (1999), Saunders (Orlando, FL), pp. 128-130. An introduction to chromatography. D. Grant, Capillary Gas Chromatography (1995), John Wiley & Sons (United Kingdom). An introduction to specialized GC. N. Grinberg, Editor, Modern Thin-Layer Chromatography (1990), Marcel Dekker (New York). Up-to-date developments in TLC. T. Hanai, HPLC: A Practical Guide (1999), Springer-Verlag (New York). HPLC introduction and applications.

E. Katz, Editor, High Performance Liquid Chromatography (1995), John Wiley & Sons (United Kingdom). A modern introduction to HPLC. A. Lehninger, D. Nelson, and M. Cox, Principles of Biochemistry, 3rd ed. (1999), Worth Publishers (New York), pp. 130-133, 384-386. Introduction to chromatography.

P. Matejtschuk, Editor, Affinity Separations: A Practical Approach (1997), IRL Press (Oxford). Principles and applications.

Further Reading

109

C. Mathews, K. van Holde, and K. Ahern, Biochemistry, 3rd ed. (2000), Benjamin/Cummings (San Francisco), pp. 150-152. Introduction to purification by chromatography. M. McMaster, HPLC: A Practical User's Guide (1994), John Wiley & Sons (New York). Very useful in the laboratory.

V. Meyer, Practical High-Performance Liquid Chromatography, 3rd ed. (1999), John Wiley & Sons (New York). Practical applications. V. Meyer, Pitfalls and Errors ofHPLC in Pictures (1998), John Wiley & Sons (New York). A useful book for the beginner. C. Morgan and N. Moir,/. Chem. Educ. 73, 1040-1041 (1996). "Rapid Microscale Isolation and Purification of Yeast Alcohol Dehydrogenase Using Cibacron Blue Affinity Chromatography." A. Niederwieser, in Methods in Enzymology, Vol. XXV, C. Hirs and S. Timasheff, Editors (1972), Academic Press (New York), pp. 60-99. Thin-layer chromatography of amino acids.

Pharmacia Biotechnology, Gel Filtration: Principles and Methods, 5th ed. (1991), Pharmacia Biotechnology, S-751 82 Uppsala, Sweden, or 800 Centennial Avenue, Piscataway, NJ 08854. An excellent guide on theory, practice, and applications of gel filtration.

R Regnier, Nature 350, 634 (1991). "Perfusion Chromatography." R. Scopes, Protein Purification: Principles and Practice, 3rd ed. (1993), Springer-Verlag (Berlin). Introduction to all methods of chromatography. J. Sherma, Anal Chem. 62, 371R-381R (1990). "Planar Chromatography."

L. Stryer, Biochemistry, 4th ed. (1995), Freeman (New York), pp. 48-50. Affinity, ionexchange, and gel-filtration chromatography. J. Touchstone, Editor, Planar Chromatography in the Life Sciences (1990), John Wiley & Sons (New York). Modern aspects of TLC. D. Voet and J. Voet, Biochemistry, 2nd ed. (1995), John Wiley & Sons (New York), pp. 78-89. Use of chromatography in protein purification. D. Voet, J. Voet, and C. Pratt, Fundamentals ofBiochemistry (1999), John Wiley & Sons (New York), pp. 99-104. Use of chromatography in protein purification. P. Williams and M. Hudson, Editors, Recent Developments in Ion Exchange (1990), Elsevier Applied Science (Amsterdam). Modern discussion of ion-exchange chromatography. Chromatography on the Web

http ://www.bio.mtu.edu/campbell/482w91 a.htm Graphical presentation of the steps in affinity chromatography. http://www.affinity-chrom.com/ Introduction and applications of affinity chromatography.

http://kerouac.pharm.uky.edu/ASRG/HPLC/hplcmytry.html A users' guide to HPLC.

Purification and Identification of Biomolecules by Chromatography

http://www.md.huji.ac.il/spectroscopy/gc.htm Principles of chromatography. Select GC, HPLC, LC, TLC

http://www.eng.rpi.eclu/clept/chem-eng/Biotech-Environ/CHROMO/chromtypes. html

Descriptions of GC, LC, Ion Exchange, and Affinity Chromatography. http://ntri.tamuk.edu/fplc/affin.html Discussion of affinity chromatography. http ://ntri.tamuk.edu/fplc/fplc 1 .html Introduction to fast performance liquid chromatography. Review Buffer preparation, Definitions of pH, Henderson-Hasselbalch equation, and Buffer calculations.

http://ultranet.eom/~jkimball/BiologyPages/A/AffinityChrom.html Application of Affinity Chromatography to purification of antibodies. Also a link to Exclusion Chromatography.

4

I Z AT I

ROTEINS y

Elect

and

NUC

Acids

phore

ctrophoresis vement of c

hniques use a to be lyze term '

al" e ec

" the ap d elec d the size, shape sep ated. Electro hnique, is capable omolecules, b

It to provide ove

widesp ing the

tofmolecul

d use in puri olecul ' es.

ool by which biochemists can examine the in an electric field. Modern electrophoretic trix as a support medium. The same medium as a spot or thin band, hence The migr ' ecules is influenced , ma of the gel support; ical composition of the molecules to is a relatively rapid and convenient nd purifying several different types of though it is diffielectrophoretic rt, zonal electrophoresis has gained ing biochemicals and in determin-

Y OF EL

The

se

nt of a ch

cted to an electric field is repre-

d by Equation 4.1.

'-? w

Equatlon 4.1

e

E

cm

4 = 111

112

Characterization of Proteins and Nucleic Acids by Electrophoresis CHAPTER 4

/= frictional coefficient, which depends on the mass and shape of the molecule

v = the velocity of the molecule

The charged particle moves at a velocity that depends directly on the electrical field (E) and charge (q), but inversely on a counteracting force generated by the viscous drag (/). The applied voltage represented by E in Equation 4.1 is usually held constant during electrophoresis, although some experiments are run under conditions of constant current (where the voltage changes with resistance) or constant power (the product of voltage and current). Under constant-voltage conditions, Equation 4.1 shows that the movement of a charged molecule depends only on the ratio q/f For molecules of similar conformation (for example, a collection of linear DNA fragments or spherical proteins),/varies with size but not shape; therefore, the only remaining variables in Equation 4.1 are the charge (q) and mass dependence of/ meaning that under such conditions molecules migrate in an electric field at a rate proportional to their charge-to-mass ratio. The movement of a charged particle in an electric field is often defined in terms of mobility, /x, the velocity per unit of electric field (Equation 4.2).

I

jjl = —

Equation 4.2

This equation can be modified using Equation 4.1. IEq

q

fji = — = —

Equation 4.3

In theory, if the net charge, q, on a molecule is known, it should be possible to measure/and obtain information about the hydrodynamic size and shape of that molecule by investigating its mobility in an electric field. Attempts to define/by electrophoresis have not been successful, primarily because Equation 4.3 does not adequately describe the electrophoretic process. Important factors that are not accounted for in the equation are interaction of migrating molecules with the support medium and shielding of the molecules by buffer ions. This means that electrophoresis is not useful for describing specific details about the shape of a molecule. Instead, it has been applied to the analysis of purity and size of macromolecules. Each molecule in a mixture is expected to have a unique charge and size, and its mobility in an electric field will therefore be unique. This expectation forms the basis for analysis and separation by all electrophoretic methods. The technique is especially useful for the analysis of amino acids, peptides, proteins, nucleotides, nucleic acids, and other charged molecules.

B.

B.

Methods of Electrophoresis

113

METHODS OF ELECTROPHORESIS

All types of electrophoresis are based on the principles just outlined. The major difference between methods is the type of support medium, which can be either cellulose or thin gels. Cellulose is used as a support medium for low-molecular-weight biochemicals such as amino acids and carbohydrates, and polyacrylamide and agarose gels are widely used as support media for larger molecules. Geometries (vertical and horizontal), buffers, and electrophoretic conditions for these two types of gels provide several different experimental arrangements, as described below. Polyacrylamide Gel Electrophoresis (PAGE)

Gels formed by polymerization of acrylamide have several positive features in electrophoresis: (1) high resolving power for small and moderately sized proteins and nucleic acids (up to approximately 1 X 106 daltons), (2) acceptance of relatively large sample sizes, (3) minimal interactions of the migrating molecules with the matrix, and (4) physical stability of the matrix. Recall from the earlier discussion of gel filtration (Chapter 3) that gels can be prepared with different pore sizes by changing the concentration of crosslinking agents. Electrophoresis through polyacrylamide gels leads to enhanced resolution of sample components because the separation is based on both molecular sieving and electrophoretic mobility. The order of molecular movement in gel filtration and PAGE is very different, however. In gel filtration (Chapter 3), large molecules migrate through the matrix faster than small molecules. The opposite is the case for gel electrophoresis, where there is no void volume in the matrix, only a continuous network of pores throughout the gel. The electrophoresis gel is comparable to a single bead in gel filtration. Therefore, large molecules do not move easily through the medium, and the rate of movement is small molecules followed by large molecules.

Polyacrylamide gels are prepared by the free radical polymerization of acrylamide and the cross-linking agent N,N}- methylene-bis-acrylamide (Figure 4.1). Chemical polymerization is controlled by an initiator-catalyst system, ammonium persulfate-TV.TV.TV'.TV'-tetramethylethylenediamine (TEMED). Photochemical polymerization may be initiated by riboflavin in the presence of ultraviolet (UV) radiation. A standard gel for protein separation is 7.5% polyacrylamide. It can be used over the molecular size range of 10,000 to 1,000,000 daltons; however, the best resolution is obtained in the range of 30,000 to 300,000 daltons. The resolving power and molecular size range of a gel depend on the concentrations of acrylamide and bis-acrylamide. Lower concentrations give gels with larger pores, allowing analysis of higher-molecular-weight biomolecules. In contrast, higher concentrations of acrylamide give gels with smaller pores, allowing analysis of lower-molecular-weight biomolecules (Table 4.1).

114

Characterization of Proteins and Nucleic Acids by Electrophoresis CHAPTER 4

Figure 4.1 Riboflavin

Ammonium persulfate

Chemical reactions illus-

trating the copolymerization of acrylamide and N,N'methylene-bis-acrylamide. See text for details.

R.

(Radicals)

CH2=CH CONhL

CONH

I CH2=CH

ChL

I

+

TEMED (catalyst)

CONH

CH2=CH

~CH2

CH

ChL

CONhL

CONH0

CH

CH

ChL

ChL

CH~ CONH

CONH

I

I

CH9

CH9

I

I

CONH

CH2

CH

CONH

CONhL

CH2

CH

CH2

CH

CH2

CH

CH2~

CONH

Polyacrylamide

Effective Range of Separation of DNA by PAGE

Acrylamide1 (% w/v) 3.5

Range of Separation (bp)

Bromophenol

Blue2

Cyanol2

1000-2000

Xylene

100

450

5.0

80-500

65

250

8.0

60-400

50

150

12.0

40-200

20

75

20.0

5-100

10

50

Ratio of acrylamide to bis-acrylamide, 20:1.

The numbers (in bp) represent the size of DNA f

ment with the same mobility as the dye.

B.

115

Meth

Polyacry ments, column

column gel. Gl acrylamide, N3 catalyst. Polym serted between tw

contains the catho

ally carried out at hence, they move d ered on top of the is also applied, whi components. Whe umn, the voltage is t stained with a dye. daily available or ca Slab gels are no which several sampl than several individ

all samples are analy sition. A typical vert acrylamide slab is p

be done using either ows the typical arrangement for a m i.d.) are filled with a mixture of ide, buffer, and free radical initiator-

o 40 minutes. The gel column is inrvoirs. The upper reservoir usually anode. Gel electrophoresis is usust biological polymers are anionic; e. The sample to be analyzed is laylied to the system. A "tracking dye" ly through the gel than the sample toved to the opposite end of the colel is removed from the column and

n gel electrophoresis are commerm inexpensive materials, d than column gels. A slab gel on is more convenient to make and use

b gels also offer the advantage that ironment that is identical in compous is shown in Figure 4.3. The polyo glass plates that are separated by

Figure 4.2

A column gel for polyaclamide electrophoresis.

Jpper buffer chamber

Sample

Pow __ supply — Gel tube

^mber

CHAPTER 4

Figure 4.3

A vertical electrophoresis apparatus for a slab gel. Courtesy of Hoefer Pharmacia Biotech, Inc., San Francisco.

spacers (Figure 4.4). The spacers allow a uniform slab thickness of 0.5 to 2.0 mm, ' ' opriate for analytical procedures. Slab gels are usually 8X1 10 cm, but for nucleotide sequencing, slab gels as large as 20 X 40 cm are often required. A plastic "co S5 ' ed into the top of the slab gel during polymerization forms inden the gel that serve as sample Is. Up to 20 sample wells may be formed. After polymerization, the comb is carefully removed and the wells are rinsed thoroughly with buffer to remove salts and any unpolymerized acrylamide. The late is clamped into place between two buffer reservoirs, a sample is lo into each is applied. For visualization, the slab is remov d stained i an te dye. Perhaps the most difficult and inconvenient aspect of polyacrylamide 1 electrophoresis is the preparation of gels. The monomer, acrylamide, is a neurotoxin and a ca ct agent; hence, special handling is required. Other gents including catalysts and initiators also require special ' d are e. In addition, it is difficult to make gels that have _„..._ le thic and compositions. Many researchers are now

B.

117

Methods of Electrophoresis

Figure 4.4

Arrangement of two glass es with spacers to form a gel. The comb is used to are wells for placement of pies.

Spacer

Back plate Comb

Front plate

turning to the use of precast now offer gels precast in glass operations are available includi gradient gel concentrations an

crylamide gels. Several manufacturers stic cassettes. Gels for all experimental gle percentage (between 3 and 27%) or riety of sample well configurations and buffer chemistries. More details on precast gels will be given in Section C, Practical Aspects of Electrophor ' . Several modifications of PA have greatly increased its versatility and usefulness as an analytical tool. Disconti in The experimental arrangem Figure 4.5. Three significant characteristics of this method are that (1) there are two gel layers, a lower or resolving gel and an upper or stacking gel; (2) the buffers used to prepare the two gel layers are of different ionic strengths and pH; and (3) the stacking gel has a lower acrylamide concentration, so its pore sizes are larger. These three changes in the experimental conditions cause the formation of highly concentrated bands of sample in the stacking gel and greater resolution of the sample components in the lower gel.

118

Characterization of Proteins and Nucleic Acids by Electrophoresis CHAPTER 4

Figure 4.5

The process of disc gel electrophoresis. A Before electrophoresis. B Movement of chloride, glycinate, and protein

through the stacking gel. C Separation of protein samples by the resolving gel.

Stacking gel

Glycine 2-3% Acrylamide pH6.9

Proteins

Chloride

Resolving gel

7.5% Acrylamide { pH8-9

> Separated proteins

Sample concentration in the upper gel occurs in the following manner. The sample is usually dissolved in glycine-chloride buffer, pH 8 to 9, before loading on the gel. Glycine exists primarily in two forms at this pH, a zwitterion and an anion (Equation 4.4). H3NCH2COO-

H2NCH2COO" + H+

Equation 4.4

B.

M

11

hods of Electrophor

The av ge ch age is turned acid sample into the upper gel, creasing the concen hence no electropho the electrophoresis s proteins and nucleic aci

8.5 is about -0.2. When the volt-

chloride) and protein or nucleic hich has a pH of 6.9. Upon entry tion 4.4 shifts toward the left, in-

on, which has no net charge and r to maintain a constant current in

s must be maintained. Since most

anionic at pH 6.9, they replace gly-

cinate as mobile ions,

ive ion mobilities in the

'

1

are chloride > protein

ample > glycinate. The

tend to accumulate and

centrated band sandwiched between

the chloride and glycin

rough the upper gel. Since the ac s low (2 to 3°/o), there is little imped-

lamide concentration in

iment to the mobility of Now, when the ioni

the glycinate concentrati most of the cu ent. The

narrow band, encounter

The increase in pH wou bility, but the smaller p ment of anions in the lo

acid sample. The separati as described in an earli

has a unique charge/ma influence its mobility. Disc gel electropho of choice for analysis of pro cleic acid bands containing the gels after electrophoresis. Sodium Dodecyi Suifate-Poly Electrophoresis (SDS-PAGE)

The electrophoretic techn* to the measurement of the m

11

molecules.

he lower gel with pH 8 to 9 buffer, anionic glycine and chloride carry eic acid sample molecules, now in a e in pH and a decrease in pore size. nd to increase electrophoretic moobility. The relative rate of movede > glycinate > protein or nucleic ponents in the resolving gel occurs lectrophoresis. Each component rete size and shape, which directly ent resolution and is the method

eic acid fragments. Protein or nuor 2 fig can be detected by staining

d@ Gel

isly discussed are not applicable hts of biological molecules beand size. If protein samples are , electrophoretic mobility then ). The molecu r weights of proto electrophoresis in the prese (SDS), and a disulfide bond

cause mobility is influenced b treated so that they have a un depends primarily on size (see teins may be estimated if they ar ence of a detergent, sodium dod hod is often called "denaturi reducing agent, mercaptoethanol electrophoresis." i SDS, the detergent disrupts When protein molecules are tr, the secondary, tertiary, and quaterna tracture to produce linear polypeptide chains coated with ne atively c ed SDS molecules. The presence of mercaptoet ol assists in protein d enaturation by reducing all disulfide

120

Characterization of Proteins and Nucleic Acids by Electrophoresis CHAPTER 4

bonds. The detergent binds to hydrophobic regions of the denatured protein chain in a constant ratio of about 1.4 g of SDS per gram of protein. The bound detergent molecules carrying negative charges mask the native charge of the protein. In essence, polypeptide chains of a constant charge/mass ratio and uniform shape are produced. The electrophoretic mobility of the SDS-protein complexes is influenced primarily by molecular size: the larger molecules are retarded by the molecular sieving effect of the gel, and the smaller molecules have greater mobility. Empirical measurements have shown a linear relationship between the log molecular weight and the electrophoretic mobility (Figure 4.6). In practice, a protein of unknown molecular weight and subunit struc ture is treated with 1% SDS and 0.1 M mercaptoethanol in electrophoresis buffer. A standard mixture of proteins with known molecular weights must also be subjected to electrophoresis under the same conditions. Two sets or standards are commercially available, one for low-molecular-weight proteins (molecular weight range 14,000 to 100,000) and one for high-molecularweight proteins (45,000 to 200,000). Figure 4.7 shows a stained gel after electrophoresis of a standard protein mixture. After electrophoresis and dye staining, mobilities are measured and molecular weights determined graphically. SDS-PAGE is valuable for estimating the molecular weight of protein subunits. This modification of gel electrophoresis finds its greatest use in characterizing the sizes and different types of subunits in oligomeric pro-

Figure 4.6

Graph illustrating the linear relationship between electrophoretic mobility of a protein and the log of its molecular weight. Thirty-seven different polypeptide chains with a molecular weight range of 11,000 to 70,000 are shown. Gels were run

in the presence of SDS (denaturing conditions). From K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969). By permission of the copyright owner, the American Society for Biochemistry and Molecular Biology, Inc.

CD

0.4

0.6

Mobility

B.

121

Methods

Figure 4.7

A silver-stained gel obtained by electrophoresis of a standard protein mixture under denaturing conditions. Samples were run on 12% polyacrylamide gels,

1

2

3

4

5

0.75 mm. Lane 1: Bovine brain

homogenate soluble fraction, 20 /jlL. Lane 2: Bovine brain homogenate soluble fraction, 10 ^L Lanes 3, 4, 5: Bio-Rad SDS-PAGE low-molecular-weight standards, three different dilu-

tions. Courtesy of Bio-Rad Laboratories, Richmond, CA.

terns. SDS-PAGE is li to a molecular weight range of 10,000 to 200,000. Gels of less t % acrylamide must be used for determini molecular weights above 200,000, but these gels do not set well and are very fragile because of cross-linking. A modification using gels of agarose-acrylamide ; allows t , measurement of mo lights above 200,000. J¥ucfe#c Acid S©qu©tsGmg G@ls

The amino acid sequence of a protein is determined by identifying amino acid residues ' red from the intact protein (see Experim t 2). Sequence analysis of nucleic acids is based on the neration

Characterization of Proteins and Nucleic Acids by Electrophoresis

of sets of DNA or RNA fragments with common ends and the separation of these oligonucleotide fragments by polyacrylamide electrophoresis. Two methods have been developed for sequencing nucleic acids: (1) the partial chemical degradation method of Maxam and Gilbert, which uses four specific chemical reactions to modify bases and cleave phosphodiester bonds, and (2) the chain termination method developed by Sanger, which requires a single-stranded DNA template and chain extension processes, followed by chain termination caused by the presence of dideoxynucleoside triphosphates. Both sequencing methods result in nested sets of DNA or RNA fragments that have one common end and chains varying in length. The smallest possible size difference of nucleic acid fragments is one nucleotide. Separation of the nucleic acid fragments by polyacrylamide electrophoresis allows one to "read" the sequence of nucleotides from the gel. The experimental arrangement is the same as that previously described for PAGE; however, the gel is prepared with many sample wells to accommodate a large number of samples. Sequence gels of 6, 8, 12, and 20% polyacrylamide are routinely used. Gels of 20% may be used to sequence the first 50 to 100 nucleotides of a nucleic acid, and lower percentage gels allow sequencing out to 250 nucleotides. Sequencing gels are large (up to 40 cm) and power supplies must provide more power than for conventional methods. Precast sequencing gels are now commercially supplied by Stratagene. They have a gel concentration of 5.5%, have 32 sample wells, and will sequence up to 500 nucleotides. Denaturants such as urea and formamide are required to prevent renaturing of the nucleic acid fragments during electrophoresis. For detection, nucleic acid chains for sequencing must be end labeled with 32P, 35S or a fluorescent tag. 32P and 35S-labeled nucleic acids on gels are detected by autoradiography (see later). Nucleic acids end labeled with fluorescent molecules are detected by fluorimeter scanning of the gels. Many researchers working on the large and expensive human genome project1 are generating huge amounts of DNA sequence data. Much of this information is stored in computer data banks for use by researchers around the world.

Agarose Gel Electrophoresis

The electrophoretic techniques discussed up to this point are useful for analyzing proteins and small fragments of nucleic acids up to 350,000 daltons (500 bp) in molecular size; however, the small pore sizes in the gel are not appropriate for analysis of large nucleic acid fragments or intact DNA molecules. The standard method used to characterize RNA and DNA in the

range 200 to 50,000 base pairs (50 kilobases) is electrophoresis with agarose as the support medium. Agarose, a product extracted from seaweed, is a linear polymer of galactopyranose derivatives. Gels are prepared by dissolving agarose in warm elec-

The human genome project is a federal government-sponsored program to sequence all DNA in human chromosomes.

B.

trophoresis bu

e to 50°C, the

is

P

lami

Gels wit

an

be

ah

on 1 arr

e to be se

is

in a s

pie well

t

fr

se

ment (Figure 4.8). wi

, and vol

'

is applied until separ

ete. Precast

of all

commercially

dlable. Nucleic acids

ing in a solution of e rescence when interc

ized on on by so bromide, a dye that displays enhanced fluoe

ed nucleic

bromide may be

ge-red bands

'c acid

The mobil'

agarose concen

ti

of the nucleic

id.

tive for nucleic

:id

the separation of D acids migrate at a r molecular weights; trophoresis results us' molecular ight. Th are superhe al cir III). The small, co st mobil'

ded, circ

trophoretic mobility of p mental conditions

tech Inc.,

n Francisco.

ses. Ethidium

ore gel formaduring electr horegels by V light re Its in

sis. Irradiation of ethi

An apparatus for horintai slab gel electrophoresis. urtesy of Hoefer Pharmacia

"

solut

tion. This me

Figure 4.8

arose sol

t.

acids ' olec

Is is influenced olecular confo

y the ation

ncentrations of 0.3 to 2.0% are most effec-

(Table 4.2). ^ iment nts on rose gels. Li ly pr to t lecular weig be

md 15 illustrate rotei ., nucleic arithm of their ated from elec-

rd nucleic acids or DNA ents of known f ons equently ered

ircu

iled rodl

II), and linear (form I molecules usually have the

inear form III molecules. The

1 mi more slowly. The relative eleci forms of D wever, depends on exncentration and ionic strength.

Characterization of Proteins and Nucleic Acids by Electrophoresis

Effective Range of Separation of DNA by Agarose

Agarose (% w/v)

Effective Range (kb)

0.3 0.5 0.7 1.2 1.5 2.0

5-50 2-25 0.8-10 0.4-5 0.2-3 0.1-2

The versatility of agarose gels is obvious when one reviews their many applications in nucleic acid analysis. The rapid advances in our understanding of nucleic acid structure and function in recent years are due primarily to the development of agarose gel electrophoresis as an analytical tool. Two of the many applications of agarose gel electrophoresis will be described here. Analysis of DNA Fragments after Digestion by Restriction Endonucleases

As described in Experiment 15, restriction endonucleases recognize a specific base sequence in double-stranded DNA and catalyze cleavage (hydrolysis of phosphodiester bonds) in or near that specific region. Many viral, bacterial, or animal DNA molecules are substrates for the enzymes. When each type of DNA is treated with a restriction endonuclease, a specific number of DNA fragments is produced. The base sequence recognized by the enzyme occurs only a few times in any particular DNA molecule; therefore, the smaller the DNA molecule, the fewer specific cleavage sites there are. Viral or phage DNA, for example, is cleaved into about 50 fragments depending on the enzyme used, whereas larger bacterial or animal DNA may be cleaved into hundreds or thousands of fragments. Smaller DNA molecules, upon cleavage with a particular enzyme, will produce a limited set of fragments. It is unlikely that this set of fragments will be the same for any two different DNA molecules, so the fragmentation pattern can be considered a "fingerprint" of the DNA substrate. The restriction pattern is produced by electrophoresis of the cleavage reaction mixture through agarose

gels, followed by staining with ethidium bromide (Figure 4.9). The separation of the fragments is based on molecular size, with large fragments remaining near the origin and smaller fragments migrating farther down the gel. In addition to characterization of DNA structure, endonuclease digestion coupled with agarose gel electrophoresis is a valuable tool for plasmid mapping (Experiment 15) and DNA recombination experiments. Characterization of Superhelical Structure of DNA

The structure of plasmid, viral, and bacterial DNA is often closed circular with negative superhelical turns. It is possible under various experimental

125



Restriction patterns produced by agarose electrophoresis of DNA fragments after restriction endonuclease action.

Courtesy of Bio-Rad Laboratories, Richmond, CA.

conditions to induce intercala DNAth

agarose in the presen pro "des mbigu and othei

conformati

n

of DNA. The

i

f supercoiled

ho is of DNA on ons of ethidium bromide en closed circular

confo

Closed circ

,

greatest electro

re

(form I) rms becau

y has the percoiled

DNA molecule

d

bromide is

d to form

e inter

DNA bas

using un-

I DNA,

winding of some of

ntration of ethid-

ium bromide is incr

e supercoils are ree conformational change electrophoresis because the moth incre >ing con tration of re rogressively u >und and This mi am repreln ' apercoils.

mo

itil no more

oft ^Asupercoil bility decreases with each ethidium bromide, the n

the electrophoretic mo se dye co (The free dye concei

sh

e related

u

erhelix den

olecule.) I

m. If more

tive superhelical turns mobility increa . For creasing trophoretic mo A

valently clo

l

i

oiling in

nt to the "re-

d DNA, posie electrophoretic conditions of in-

l decrease in elecon r

ge.

isomers of native, co-

r degr

supercoiltion of

126

Characterization of Proteins and Nucleic Acids by Electrophoresis CHAPTER 4

enzymes that catalyze changes in the conformation or topology of native DNA. These enzymes, called topoisomerases, have been isolated from bacterial and mammalian cells. They change DNA conformations by catalyzing nicking and closing of phosphodiester bonds in circular duplex DNA. Agarose gel electrophoresis is an ideal method for identifying and assaying topoisomerases because the intermediate DNA molecules can be resolved on the basis of the extent of supercoiling. Topoisomerases may be assayed by incubating native DNA with an enzyme preparation, removing aliquots after various periods of time, and subjecting them to electrophoresis on an agarose gel with standard supercoiled and relaxed DNA. Pulsed Field Gel Electrophoresis (PFGE)

Conventional agarose gel electrophoresis is limited in use for the separation of nucleic acid fragments smaller than 50,000 bp (50 kb). In practice, that limit is closer to 20,000 to 30,000 bp if high resolution is desired. Since chromosomal DNA from most organisms contains thousands and even millions of base pairs, the DNA must be cleaved by restriction enzymes before analysis by standard electrophoresis. In the early 1980s it was discovered by Schwartz and Cantor at Columbia University that large molecules of DNA (yeast chromosomes, 200-3000 kb) could be separated by pulsed field gel electrophoresis (PFGE). There is one major distinction between standard gel electrophoresis and PFGE. In PFGE, the electric field is not constant as in the standard method but is changed repeatedly (pulsed) in direction and strength during the separation (Figure 4.10). The physical mechanism for

Sample well

^

dnaO7 fragments

Field direction

Gel